Two hybrid assay that detects HIV-1 reverse transcriptase dimerization

ABSTRACT

This invention provides methods of determining whether a compound inhibits HIV-1 reverse transcriptase. This invention provides methods of determining whether a compound inhibits formation of a complex between a p66 and p51 subunit polypeptides of HIV-1 reverse transcriptase. This invention provides a method of determining whether a compound enhances formation of a complex between a p66 and p51 subunit polypeptides of HIV-1 reverse transcriptase. This invention provides methods of determining whether a compound inhibits formation of a complex between two p66 subunit polypeptides of HIV-1 reverse transcriptase. This invention provides methods of determining whether a compound enhances formation of a complex between two p66 subunit polypeptides of HIV-1 reverse transcriptase.

This invention is a continuation-in-part and claims the benefit of U.S.Ser. No. 09/588,939, filed Jun. 6, 2000, now abandoned the contents ofwhich are hereby incorporated by reference into this application.

The invention disclosed herein was made in part with Government supportunder NIH Grant No. AI 27690. Accordingly, the government has certainrights in this invention.

Throughout this application, various publications are referenced withinparentheses. Disclosures of these publications in their entireties arehereby incorporated by reference into this application to more fullydescribe the state of the art to which this invention pertains. Fullbibliographic citations for these references may be found immediatelypreceding the claims.

BACKGROUND OF THE INVENTION

HIV-1 reverse transcriptase (RT) catalyzes the conversion of genomic RNAinto cDNA. The enzyme is a heterodimer of p66 and p51 subunits, and thedimerization of these subunits is required for optimal enzyme activity.To analyze this process at the genetic level we developed constructsthat permit the detection of the interaction between these subunits inthe yeast two-hybrid system. Genetic analysis of RT subdomains requiredfor heterodimerization revealed that the fingers and palm of p66 weredispensable for p51 interaction. However, as little as a 26-amino aciddeletion at the C terminus of p51 prevented dimerization with p66. Aprimer grip mutation, L234A, previously shown to inhibit RT dimerizationby biochemical assays, also prevented RT dimerization in the yeasttwo-hybrid system. Second-site mutations that restored RT dimerizationin yeast to the L234A parent were recovered in the tryptophan repeatregion at the dimer interface and at the polymerase active site,suggesting the involvement of these sites in RT dimerization. In vitrobinding experiments confirmed the effects of the L234A mutation and thesuppressor mutations on the interaction of the two subunits. The RTtwo-hybrid assay should facilitate the extensive genetic analysis of RTdimerization and should make possible the rapid screening of potentialinhibitors of this essential process.

The HIV type 1 (HIV-1) reverse transcriptase (RT) is required for theconversion of genomic RNA into double-stranded proviral DNA, catalyzedby the RNA- and DNA-dependent polymerase and ribonuclease H activitiesof the enzyme. HIV-1 RT is an asymmetric dimer formed by the associationof p66 and p51 polypeptides, which are cleaved from a largePr160^(GagPol) precursor by the viral protease during virion assemblyp51 contains identical N-terminal sequences as p66, but lacks theC-terminal ribonuclease H (RNase H) domain (1). The structure of HIV-1RT has been elucidated by x-ray crystallography in a variety ofconfigurations, including unliganded (2), complexed to nonnucleoside RTinhibitors (3), or complexed with double-stranded DNA either with (4) orwithout deoxynucleotide triphosphate (5, 6). Such analyses have shownthat p66 can be divided structurally into the polymerase and RNase Hdomains, with the polymerase domain further divided into the fingers,palm, thumb and connections subdomains (6). Although p51 has the samepolymerase domains as p66, the relative orientations of these individualdomains differ markedly, resulting in p51 assuming a closed structure.

The RT heterodimer represents the biologically relevant form of theenzyme; the monomeric subunits have only low catalytic activity (7).Structural analysis reveals three major contacts between p66 and p51,with most of the interaction surfaces being largely hydrophobic (8, 9).The three contacts comprise an extensive dimer interface that includesthe fingers subdomain of p51 with the palm of p66, the connectionsubdomains of both subunits, and the thumb subdomain of p51 with theRNase H domain of p66 (9). Several single amino acid substitutions inHIV-1 RT have been shown to inhibit heterodimer association (10-12).These include the mutations L234A (10, 11), G231A (11) and W229A (11),all located in the primer grip region of the p66 subunit, and L289K (12)in the thumb subdomain. Remarkably, these mutations are not located atthe dimer interface and probably mediate their effects indirectlythrough conformational changes in the p66 subunit.

Several biochemical assays have been used previously to specificallymeasure RT dimerization. Some are based on the physical separation ofmonomers and dimers as determined by analytical ultracentrifugation (8)and gel filtration (7). Other assays include intrinsic tryptophanfluorescence (13) chemical crosslinking (14), the use of affinity tags(15) and polymerase activity itself (7). Although these methods detectdimerization, they either lack specificity or are not easy to perform.Moreover, these assays do not facilitate the rapid genetic analysis ofprotein-protein interactions under physiological conditions nor are theysuitable for high throughput screening for RT dimerization inhibitors.

The yeast two-hybrid (Y2H) system (16) has been exploited to study thehomomeric interactions of several retroviral proteins (see, e.g., ref.17), and heteromeric interactions between viral proteins and variouscellular partners (see, e.g., ref. 18). We have used this system toperform a genetic analysis of the determinants of RT dimerization. Inaddition, we have identified second-site mutations that restoreheterodimerization to a noninteracting mutant p66.

SUMMARY OF THE INVENTION

This invention provides a method of determining whether a compoundinhibits HIV-1 reverse transcriptase which comprises:

a) contacting a yeast cell with the compound, which cell comprises (i) afirst plasmid which expresses a fusion protein comprising a p66 subunitpolypeptide of HIV-1 reverse transcriptase, (ii) a second plasmid whichexpresses a fusion protein comprising a p51 subunit polypeptide of HIV-1reverse transcriptase, and (iii) a reporter gene which is activated inthe presence of a complex between the p66 subunit polypeptide and thep51 subunit polypeptide, and determining the level of activity of thereporter gene in the cell in the presence of the compound; and

b) comparing the level of activity of the reporter gene determined instep (a) with a level of activity of the reporter gene determined in theabsence of the compound, wherein a decreased level of activity of thereporter gene in step (a) indicates that the compound inhibits formationof a complex between the p51 subunit polypeptide of HIV-1 reversetranscriptase and the p66 subunit polypeptide of HIV-1 reversetranscriptase, thereby indicating that the compound inhibits HIV-1reverse transcriptase.

This invention provides a method of determining whether a compoundinhibits formation of a complex between a p66 subunit polypeptide ofHIV-1 reverse transcriptase and a p51 subunit polypeptide of HIV-1reverse transcriptase which comprises:

a) contacting a yeast cell with the compound, which cell comprises (i) afirst plasmid which expresses a fusion protein comprising a p66 subunitpolypeptide of HIV-1 reverse transcriptase, (ii) a second plasmid whichexpresses a fusion protein comprising a p51 subunit polypeptide of HIV-1reverse transcriptase, and (iii) a reporter gene which is activated inthe presence of a complex between the p66 subunit polypeptide and thep51 subunit polypeptide, and determining the level of activity of thereporter gene in the cell in the presence of the compound; and

b) comparing the level of activity of the reporter gene determined instep (a) with a level of activity of the reporter gene determined in theabsence of the compound, wherein a decreased level of activity of thereporter gene in step (a) indicates that the compound inhibits formationof a complex between the p51 subunit polypeptide of HIV-1 reversetranscriptase and the p66 subunit polypeptide of HIV-1 reversetranscriptase.

This invention provides a method of determining whether a compoundinhibits HIV-1 reverse transcriptase which comprises:

a) contacting a yeast cell with the compound, which cell comprises (i) afirst plasmid which expresses a fusion protein comprising a p66 subunitpolypeptide of HIV-1 reverse transcriptase, (ii) a second plasmid whichexpresses a fusion protein comprising a p51 subunit polypeptide of HIV-1reverse transcriptase, and (iii) a reporter gene which is activated inthe presence of a complex between the p66 subunit polypeptide and thep51 subunit polypeptide, and determining the level of activity of thereporter gene in the cell in the presence of the compound; and

b) comparing the level of activity of the reporter gene determined instep (a) with a level of activity of the reporter gene determined in theabsence of the compound, wherein an increased level of activity of thereporter gene determined in step (a) indicates that the compound is anactivator of the formation of the complex between the p51 subunitpolypeptide of HIV-1 reverse transcriptase and the p66 subunitpolypeptide of HIV-1 reverse transcriptase, thereby indicating that thecompound inhibits HIV-1 reverse transcriptase.

This invention provides a method of determining whether a compoundenhances formation of a complex between a p66 subunit polypeptide ofHIV-1 reverse transcriptase and a p51 subunit polypeptide of HIV-1reverse transcriptase which comprises:

a) contacting a yeast cell with the compound, which cell comprises (i) afirst plasmid which expresses a fusion protein comprising a p66 subunitpolypeptide of HIV-1 reverse transcriptase, (ii) a second plasmid whichexpresses a fusion protein comprising a p51 subunit polypeptide of HIV-1reverse transcriptase, and (iii) a reporter gene which is activated inthe presence of a complex between the p66 subunit polypeptide and thep51 subunit polypeptide, and determining the level of activity of thereporter gene in the cell in the presence of the compound; and

b) comparing the level of activity of the reporter gene determined instep (a) with a level of activity of the reporter gene determined in theabsence of the compound, wherein an increased level of activity of thereporter gene determined in step (a) indicates that the compound is anactivator of the formation of the complex between the p51 subunitpolypeptide of HIV-1 reverse transcriptase and the p66 subunitpolypeptide of HIV-1 reverse transcriptase.

This invention provides a method of determining whether a compoundinhibits HIV-1 reverse transcriptase which comprises:

a) contacting a yeast cell with the compound, which cell comprises (i) afirst plasmid which expresses a fusion protein comprising a first p66subunit polypeptide of HIV-1 reverse transcriptase, (ii) a secondplasmid which expresses a fusion protein comprising a second p66 subunitpolypeptide of HIV-1 reverse transcriptase, and (iii) a reporter genewhich is activated in the presence of a complex between the first p66subunit polypeptide and the second p66 subunit polypeptide, anddetermining the level of activity of the reporter gene in the cell inthe presence of the compound; and

b) comparing the level of activity of the reporter gene determined instep (a) with a level of activity of the reporter gene determined in theabsence of the compound, wherein a decreased level of activity of thereporter gene in step (a) indicates that the compound inhibits formationof a complex between the first p66 subunit polypeptide of HIV-1 reversetranscriptase and the second p66 subunit polypeptide of HIV-1 reversetranscriptase, thereby indicating that the compound inhibits HIV-1reverse transcriptase.

This invention provides a method of determining whether a compoundinhibits formation of a complex between a first p66 subunit polypeptideof HIV-1 reverse transcriptase and a second p66 subunit polypeptide ofHIV-1 reverse transcriptase which comprises:

a) contacting a yeast cell with the compound, which cell comprises (i) afirst plasmid which expresses a fusion protein comprising a first p66subunit polypeptide of HIV-1 reverse transcriptase, (ii) a secondplasmid which expresses a fusion protein comprising a second p66 subunitpolypeptide of HIV-1 reverse transcriptase, and (iii) a reporter genewhich is activated in the presence of a complex between the first p66subunit polypeptide and the second p66 subunit polypeptide, anddetermining the level of activity of the reporter gene in the cell inthe presence of the compound; and

b) comparing the level of activity of the reporter gene determined instep (a) with a level of activity of the reporter gene determined in theabsence of the compound, wherein a decreased level of activity of thereporter gene in step (a) indicates that the compound inhibits formationof a complex between the first p66 subunit polypeptide of HIV-1 reversetranscriptase and the second p66 subunit polypeptide of HIV-1 reversetranscriptase.

This invention provides a method of determining whether a compoundinhibits HIV-1 reverse transcriptase which comprises:

a) contacting a yeast cell with the compound, which cell comprises (i) afirst plasmid which expresses a fusion protein comprising a first p66subunit polypeptide of HIV-1 reverse transcriptase, (ii) a secondplasmid which expresses a fusion protein comprising a second p66 subunitpolypeptide of HIV-1 reverse transcriptase, and (iii) a reporter genewhich is activated in the presence of a complex between the first p66subunit polypeptide and the second p66 subunit polypeptide, anddetermining the level of activity of the reporter gene in the cell inthe presence of the compound; and

b) comparing the level of activity of the reporter gene determined instep (a) with a level of activity of the reporter gene determined in theabsence of the compound, wherein an increased level of activity of thereporter gene in step (a) indicates that the compound is an activator ofthe formation of the complex between the first p66 subunit polypeptideof HIV-1 reverse transcriptase and the second p66 subunit polypeptide ofHIV-1 reverse transcriptase, thereby indicating that the compoundinhibits HIV-1 reverse transcriptase.

This invention provides a method of determining whether a compoundenhances formation of a complex between a first p66 subunit polypeptideof HIV-1 reverse transcriptase and a second p66 subunit polypeptide ofHIV-1 reverse transcriptase which comprises:

a) contacting a yeast cell with the compound, which cell comprises (i) afirst plasmid which expresses a fusion protein comprising a first p66subunit polypeptide of HIV-1 reverse transcriptase, (ii) a secondplasmid which expresses a fusion protein comprising a second p66 subunitpolypeptide of HIV-1 reverse transcriptase, and (iii) a reporter genewhich is activated in the presence of a complex between the first p66subunit polypeptide and the second p66 subunit polypeptide, anddetermining the level of activity of the reporter gene in the cell inthe presence of the compound; and

b) comparing the level of activity of the reporter gene determined instep (a) with a level of activity of the reporter gene determined in theabsence of the compound, wherein an increased level of activity of thereporter gene in step (a) indicates that the compound is an activator ofthe formation of the complex between the first p66 subunit polypeptideof HIV-1 reverse transcriptase and the second p66 subunit polypeptide ofHIV-1 reverse transcriptase, thereby indicating that the compoundinhibits HIV-1 reverse transcriptase.

This invention provides a method of making a pharmaceutical compositionwhich comprises:

a) determining whether a compound inhibits HIV-1 reverse transcriptaseby one of the methods described herein;

b) recovering the compound if it is determined to inhibit HIV-1 reversetranscriptase; and

c) admixing the compound with a pharmaceutically acceptable carrier.

This invention provides a method of inhibiting formation of a complexbetween the p51 subunit polypeptide of HIV-1 reverse transcriptase and ap66 subunit polypeptide of HIV-1 reverse transcriptase, which comprisescontacting either (1) the p51 subunit polypeptide, (2) the p66 subunitpolypeptide, or (3) both the p51 subunit polypeptide and the p66 subunitpolypeptide, with an effective amount of a compound determined to do soby the method of claim 2, so to thereby inhibit formation of a complexbetween the p51 subunit polypeptide of HIV-1 reverse transcriptase and ap66 subunit polypeptide of HIV-1 reverse transcriptase.

This invention provides a method of enhancing formation of a complexbetween the p51 subunit polypeptide of HIV-1 reverse transcriptase and ap66 subunit polypeptide of HIV-1 reverse transcriptase, which comprisescontacting either (1) the p51 subunit polypeptide, (2) the p66 subunitpolypeptide, or (3) both the p51 subunit polypeptide and the p66 subunitpolypeptide, with an effective amount of a compound determined to do soby the method of claim 4, so to thereby enhance formation of a complexbetween the p51 subunit polypeptide of HIV-1 reverse transcriptase and ap66 subunit polypeptide of HIV-1 reverse transcriptase.

This invention provides a method of inhibiting formation of a complexbetween a first p66 subunit polypeptide of HIV-1 reverse transcriptaseand a second p66 subunit polypeptide of HIV-1 reverse transcriptase,which comprises contacting either (1) the first p66 subunit polypeptide,(2) the second p66 subunit polypeptide, or (3) both the first p66subunit polypeptide and the second p66 subunit polypeptide, with aneffective amount of a compound determined to do so by the method ofclaim 6, so to thereby inhibit formation of a complex between the firstp66 subunit polypeptide of HIV-1 reverse transcriptase and the secondp66 subunit polypeptide of HIV-1 reverse transcriptase.

This invention provides a method of enhancing formation of a complexbetween a first p66 subunit polypeptide of HIV-1 reverse transcriptaseand a second p66 subunit polypeptide of HIV-1 reverse transcriptase,which comprises contacting either (1) the first p66 subunit polypeptide,(2) the second p66 subunit polypeptide, or (3) both the first p66subunit polypeptide and the second p66 subunit polypeptide, with aneffective amount of a compound determined to do so by the method ofclaim 8, so to thereby enhance formation of a complex between the firstp66 subunit polypeptide of HIV-1 reverse transcriptase and the secondp66 subunit polypeptide of HIV-1 reverse transcriptase.

This invention provides a compound determined to be capable ofinhibiting formation of a complex between a p51 subunit polypeptide ofHIV-1 reverse transcriptase and a p66 subunit polypeptide of HIV-1reverse transcriptase.

This invention provides a compound determined to be capable of enhancingformation of a complex between a p51 subunit polypeptide of HIV-1reverse transcriptase and a p66 subunit polypeptide of HIV-1 reversetranscriptase.

This invention provides a compound determined to be capable ofinhibiting formation of a complex between a first p66 subunitpolypeptide of HIV-1 reverse transcriptase and a second p66 subunitpolypeptide of HIV-1 reverse transcriptase.

This invention provides a compound determined to be capable of enhancingformation of a complex between a first p66 subunit polypeptide of HIV-1reverse transcriptase and a second p66 subunit polypeptide of HIV-1reverse transcriptase.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1

RT fusion constructs, encoded fusion proteins and expression of fusionsin yeast reporter strains. The six-alanine linker is denoted by thehatched box, and the HA epitope by black shaded regions, p66 and p51indicate the 66 kDa and 51 kDa subunits of HIV-1 RT, respectively.Expression of fusion proteins was determined by introducing theindicated plasmids into CTY10-5d, except for p66 GBT9 and p51AS2-1 whichwere introduced into HF7c. Fusion protein expression was detected byprobing yeast protein lysates with anti-RT antibodies as described inthe Materials and Methods. ++, high; +, moderate; +/−, low and −,undetectable protein expression. ND, not done.

FIG. 2

Interaction of p66 deletion mutants with Gal4AD-HA-51 fusion protein,p66 polymerase domains were fused to the C-terminus of lexA87 in pSH2-1.CTY10-5d was cotransformed with the appropriate constructs.Transformants were lifted onto nitrocellulose and subjected to β-galcolony lift assay to determine intensities of blue color produced asdefined in Tables 1 and 2. β-gal activity from liquid assays isexpressed in Miller Units. Expression in CTY10-5d of p66 fusion proteinswas detected using anti-lexA polyclonal antibodies. Expression levelsare as defined as in the legend for FIG. 1.

FIG. 3

Interaction of C-terminal deletion mutants of p51 with lexA202-Ala-66,p51 domains were fused to the C-terminus of the Gal4AD in pACTII.Deletions at the C-terminus are denoted by the number of amino acidsmissing from the end of p51. β-gal activity was determined as describedin the legend of FIG. 2. Expression of p51 fusion proteins in CTY10-5dwas detected using anti-GAL4AD antibodies, and expression levels are asdenoted in the legend for FIG. 1.

FIG. 4

L234A inhibits RT dimerization in the Y2H assay. CTY10-5d wascotransformed with expression constructs, and yeast patches weresubjected to both the β-gal colony lift and liquid assays. The green ishydrolyzed X-gal and reflects β-gal activity. p66 wt and p51 wt denotewild-type lex202-Ala-HX66 and Gal4AD-HX51 fusion proteins, respectively.pAD denotes pGADNOT. p66mut and p51mut denote RT fusion proteinslex202-Ala-66-L234A and Gal4AD-51-L234A, respectively.

FIG. 5

Ribbon diagram of unliganded HIV-1 RT showing position of L234A primergrip mutation and locations of suppressors (shaded black). The figurewas generated by MOLSCRIPT (38) and RASTER3D (39) with coordinates (2)retrieved from the Research Collaboratory for Structural Bioinformatics(RCSB) Protein Data Bank (PDB) (http://www.rcsb.org/pdb, PDB ID:lHMV.pdb). Domains are defined as in (3) with fingers, blue; palm,green; thumb, yellow; connection, red; and RNase H in purple. Domains inp66 are in fully saturated colors, whereas in p51 they have decreasedsaturation. Secondary structure was assigned using DSSP (40). Spiralsrepresent alpha-helices, arrows denote beta-strands.

FIG. 6

In vitro assay for binding of GST-p51 and p66 to form active RTheterodimers. Panel A: Bacterial lysates containing GST-p51 and variousp66 proteins as indicated were incubated overnight and captured onglutathione beads. The complexes were eluted, resolved by SDS-PAGE,blotted to membrane and detected by monoclonal anti-RT antibodies. Mock,GST-p51 alone. Panel B: An aliquot of each incubation mix, reflectinginput protein, was directly analyzed by SDS-PAGE and Western blot as inPanel A. Panel C: Bound proteins were eluted with glutathione andassayed for RT activity with homopolymeric template-primer. Values arenormalized to the wild-type control.

FIG. 7

Dose-response curve showing the enhancement by NNRTIs of b-gal activityin yeast cotransformed with lexA₈₇-66 and Gal4AD-51. The fold increasein b-gal activity was calculated by dividing b-gal activity (in MillerUnits) for each drug concentration with the b-gal activity from cellsgrown in the absence of inhibitor. The data represents the averageresults from two independent experiments. The concentration of drug thatmediates a 5-fold increase in b-gal activity is shown in parenthesis. A:b-gal enhancement activity of the NNRTIs, efavirenz, HBY 097, a-APA,nevirapine, 8-Cl-TIBO and delavirdine. B: b-gal enhancement activity ofthe carboxanilide class of NNRTIs.

FIG. 8

Effect of the Y181C mutation on enhancement of b-gal activity in yeastby nevirapine. Yeast expressing wild-type lexA₈₇-66 and Gal4AD-51 ormutant lexA₈₇-66Y181C and wild type Gal4AD-51 were grown in the presenceof nevirapine and assayed for b-gal activity. Results are expressed asfold increase in b-gal activity compared to untreated cells. Values ontop of each bar indicates b-gal activity (in Miller Units) +/−standarddeviation.

FIG. 9

Effect of efavirenz on b-gal activity in yeast expressing thedimerization defective mutants L234A and W401A. Yeast expressingwild-type p66 bait and p51 prey fusions, mutant p66 bait and wild-typep51 prey and mutant p66 bait and mutant p51 prey fusions were assayedfor b-gal activity. Results are expressed as the fold increase in b-galactivity compared to untreated controls. Values on top of each barindicates b-gal activity in Miller Units. Effect of efavirenz on yeastexpressing bait and prey fusions with the W401A change (A) or L234Achange (B).

FIG. 10

Coimmunoprecipitaton assay detecting heterodimer formation in yeastpropagated in the presence of NNRTIs. (A): Yeast expressing p66 bait andp51 prey fusions containing the W401A mutation were grown in thepresence of efavirenz (EFV), UC781 or no drug. After growth, yeast wereprocessed in the absence or presence of added drug (drug in ip).Heterodimers present in lysates were detected by immunoprecipation ofGal4AD-HA-51W401A with anti-HA antibodies followed by immunodetection ofcoimmunoprecipitated p66. The b-gal activity for each treatment wasdetermined and expressed in Miller Units. (B). Yeast expressing p66 baitand p51 prey fusions containing the L234A mutation.

FIG. 11

Western blot analysis of HIV-1 RT heterodimers formed in the presence ofefavirenz (EFV) in vitro. Bacteria expressing either wild-type p66-Hisand GST-p51, or dimerization defective mutants were induced and lysateswere prepared. Lysates were mixed and incubated overnight at 4° C. withor without drug and dimers were captured by binding to GlutathioneSepharose 4B beads. Heterodimer bound to beads were resolved by SDS-PAGEand proteins detected by probing with anti-RT monoclonal antibodies.

FIG. 12

Western blot analysis of HIV-1 RT heterodimers formed in the presence ofNNRTIs in vitro. Bacterially expressed proteins p66-His and GST-p51 werecombined in the presence of 1-1000 fold molar excess of drug andincubated overnight. Heterodimers were captured and detected asdescribed in the legend of FIG. 11.

FIG. 13

Western blot analysis of heterodimer formation after pretreatment of oneof the subunits with efavirenz. p66-His, GST-p51 and M15 bacteriallysate were preincubated in the absence or presence of 10-1000 foldmolar excess of efavirenz. Lysates were washed and the presence ofresidual efavirenz was assayed by the addition of GST-p51, p66-His orboth subunits, respectively. Heterodimers were captured and detected asdescribed in the legend of FIG. 11.

FIG. 14

Molecular surface representation of the p66 and p51 subunits of HIV-1RT. Residues colored yellow (p66) or magenta (p51) are amino acids thatare not accessible to solvent in the presence of the other subunit inthe heterodimeric form. The NNRTI binding pocket is shown in red. Thesum of the surface areas colored in yellow and magenta is the totalburied surface area at the interface of the two subunits.

FIG. 15

Binding of delavirdine (BHAP) (A) and UC781 (B) at the interface of thep66 (magenta) and p51 (yellow) subunits of the HIV-1 RT. Delavirdine, alarge inhibitor, is bound further away from the p66/p51 interface. Therelative orientation of the inhibitors in the NNRTI binding pocket isshown in (C). Some residues that comprise the NNRTI binding site havebeen omitted for clarity.

DETAILED DESCRIPTION OF THE INVENTION

This invention provides a method of determining whether a compoundinhibits HIV-1 reverse transcriptase which comprises:

a) contacting a yeast cell with the compound, which cell comprises (i) afirst plasmid which expresses a fusion protein comprising a p66 subunitpolypeptide of HIV-1 reverse transcriptase, (ii) a second plasmid whichexpresses a fusion protein comprising a p51 subunit polypeptide of HIV-1reverse transcriptase, and (iii) a reporter gene which is activated inthe presence of a complex between the p66 subunit polypeptide and thep51 subunit polypeptide, and determining the level of activity of thereporter gene in the cell in the presence of the compound; and

b) comparing the level of activity of the reporter gene determined instep (a) with a level of activity of the reporter gene determined in theabsence of the compound, wherein a decreased level of activity of thereporter gene in step (a) indicates that the compound inhibits formationof a complex between the p51 subunit polypeptide of HIV-1 reversetranscriptase and the p66 subunit polypeptide of HIV-1 reversetranscriptase, thereby indicating that the compound inhibits HIV-1reverse transcriptase.

This invention provides a method of determining whether a compoundinhibits formation of a complex between a p66 subunit polypeptide ofHIV-1 reverse transcriptase and a p51 subunit polypeptide of HIV-1reverse transcriptase which comprises:

a) contacting a yeast cell with the compound, which cell comprises (i) afirst plasmid which expresses a fusion protein comprising a p66 subunitpolypeptide of HIV-1 reverse transcriptase, (ii) a second plasmid whichexpresses a fusion protein comprising a p51 subunit polypeptide of HIV-1reverse transcriptase, and (iii) a reporter gene which is activated inthe presence of a complex between the p66 subunit polypeptide and thep51 subunit polypeptide, and determining the level of activity of thereporter gene in the cell in the presence of the compound; and

b) comparing the level of activity of the reporter gene determined instep (a) with a level of activity of the reporter gene determined in theabsence of the compound, wherein a decreased level of activity of thereporter gene in step (a) indicates that the compound inhibits formationof a complex between the p51 subunit polypeptide of HIV-1 reversetranscriptase and the p66 subunit polypeptide of HIV-1 reversetranscriptase.

This invention provides a method of determining whether a compoundinhibits HIV-1 reverse transcriptase which comprises:

a) contacting a yeast cell with the compound, which cell comprises (i) afirst plasmid which expresses a fusion protein comprising a p66 subunitpolypeptide of HIV-1 reverse transcriptase, (ii) a second plasmid whichexpresses a fusion protein comprising a p51 subunit polypeptide of HIV-1reverse transcriptase, and (iii) a reporter gene which is activated inthe presence of a complex between the p66 subunit polypeptide and thep51 subunit polypeptide, and determining the level of activity of thereporter gene in the cell in the presence of the compound; and

b) comparing the level of activity of the reporter gene determined instep (a) with a level of activity of the reporter gene determined in theabsence of the compound, wherein an increased level of activity of thereporter gene determined in step (a) indicates that the compound is anactivator of the formation of the complex between the p51 subunitpolypeptide of HIV-1 reverse transcriptase and the p66 subunitpolypeptide of HIV-1 reverse transcriptase, thereby indicating that thecompound inhibits HIV-1 reverse transcriptase.

This invention provides a method of determining whether a compoundenhances formation of a complex between a p66 subunit polypeptide ofHIV-1 reverse transcriptase and a p51 subunit polypeptide of HIV-1reverse transcriptase which comprises:

a) contacting a yeast cell with the compound, which cell comprises (i) afirst plasmid which expresses a fusion protein comprising a p66 subunitpolypeptide of HIV-1 reverse transcriptase, (ii) a second plasmid whichexpresses a fusion protein comprising a p51 subunit polypeptide of HIV-1reverse transcriptase, and (iii) a reporter gene which is activated inthe presence of a complex between the p66 subunit polypeptide and thep51 subunit polypeptide, and determining the level of activity of thereporter gene in the cell in the presence of the compound; and

b) comparing the level of activity of the reporter gene determined instep (a) with a level of activity of the reporter gene determined in theabsence of the compound, wherein an increased level of activity of thereporter gene determined in step (a) indicates that the compound is anactivator of the formation of the complex between the p51 subunitpolypeptide of HIV-1 reverse transcriptase and the p66 subunitpolypeptide of HIV-1 reverse transcriptase.

This invention provides a method of determining whether a compoundinhibits HIV-1 reverse transcriptase which comprises:

a) contacting a yeast cell with the compound, which cell comprises (i) afirst plasmid which expresses a fusion protein comprising a first p66subunit polypeptide of HIV-1 reverse transcriptase, (ii) a secondplasmid which expresses a fusion protein comprising a second p66 subunitpolypeptide of HIV-1 reverse transcriptase, and (iii) a reporter genewhich is activated in the presence of a complex between the first p66subunit polypeptide and the second p66 subunit polypeptide, anddetermining the level of activity of the reporter gene in the cell inthe presence of the compound; and

b) comparing the level of activity of the reporter gene determined instep (a) with a level of activity of the reporter gene determined in theabsence of the compound, wherein a decreased level of activity of thereporter gene in step (a) indicates that the compound inhibits formationof a complex between the first p66 subunit polypeptide of HIV-1 reversetranscriptase and the second p66 subunit polypeptide of HIV-1 reversetranscriptase, thereby indicating that the compound inhibits HIV-1reverse transcriptase.

This invention provides a method of determining whether a compoundinhibits formation of a complex between a first p66 subunit polypeptideof HIV-1 reverse transcriptase and a second p66 subunit polypeptide ofHIV-1 reverse transcriptase which comprises:

a) contacting a yeast cell with the compound, which cell comprises (i) afirst plasmid which expresses a fusion protein comprising a first p66subunit polypeptide of HIV-1 reverse transcriptase, (ii) a secondplasmid which expresses a fusion protein comprising a second p66 subunitpolypeptide of HIV-1 reverse transcriptase, and (iii) a reporter genewhich is activated in the presence of a complex between the first p66subunit polypeptide and the second p66 subunit polypeptide, anddetermining the level of activity of the reporter gene in the cell inthe presence of the compound; and

b) comparing the level of activity of the reporter gene determined instep (a) with a level of activity of the reporter gene determined in theabsence of the compound, wherein a decreased level of activity of thereporter gene in step (a) indicates that the compound inhibits formationof a complex between the first p66 subunit polypeptide of HIV-1 reversetranscriptase and the second p66 subunit polypeptide of HIV-1 reversetranscriptase.

This invention provides a method of determining whether a compoundinhibits HIV-1 reverse transcriptase which comprises:

a) contacting a yeast cell with the compound, which cell comprises (i) afirst plasmid which expresses a fusion protein comprising a first p66subunit polypeptide of HIV-1 reverse transcriptase, (ii) a secondplasmid which expresses a fusion protein comprising a second p66 subunitpolypeptide of HIV-1 reverse transcriptase, and (iii) a reporter genewhich is activated in the presence of a complex between the first p66subunit polypeptide and the second p66 subunit polypeptide, anddetermining the level of activity of the reporter gene in the cell inthe presence of the compound; and

b) comparing the level of activity of the reporter gene determined instep (a) with a level of activity of the reporter gene determined in theabsence of the compound, wherein an increased level of activity of thereporter gene in step (a) indicates that the compound is an activator ofthe formation of the complex between the first p66 subunit polypeptideof HIV-1 reverse transcriptase and the second p66 subunit polypeptide ofHIV-1 reverse transcriptase, thereby indicating that the compoundinhibits HIV-1 reverse transcriptase.

This invention provides a method of determining whether a compoundenhances formation of a complex between a first p66 subunit polypeptideof HIV-1 reverse transcriptase and a second p66 subunit polypeptide ofHIV-1 reverse transcriptase which comprises:

a) contacting a yeast cell with the compound, which cell comprises (i) afirst plasmid which expresses a fusion protein comprising a first p66subunit polypeptide of HIV-1 reverse transcriptase, (ii) a secondplasmid which expresses a fusion protein comprising a second p66 subunitpolypeptide of HIV-1 reverse transcriptase, and (iii) a reporter genewhich is activated in the presence of a complex between the first p66subunit polypeptide and the second p66 subunit polypeptide, anddetermining the level of activity of the reporter gene in the cell inthe presence of the compound; and

b) comparing the level of activity of the reporter gene determined instep (a) with a level of activity of the reporter gene determined in theabsence of the compound, wherein an increased level of activity of thereporter gene in step (a) indicates that the compound is an activator ofthe formation of the complex between the first p66 subunit polypeptideof HIV-1 reverse transcriptase and the second p66 subunit polypeptide ofHIV-1 reverse transcriptase, thereby indicating that the compoundinhibits HIV-1 reverse transcriptase.

The methods described herein may also be adapted to other types of cellsin addition to a yeast cell. Other cell types include but are notlimited to eucaryotic, procaryotic, bacteria, E. coli, mammalian andhuman cells.

In one embodiment of the methods described herein, (a) the fusionprotein expressed by the first plasmid comprises a peptide having a DNAbinding domain, and (b) the fusion protein expressed by the secondplasmid comprises a peptide having a transcription activation domain.

In one embodiment of the methods described herein, (a) the fusionprotein expressed by the first plasmid comprises a peptide having atranscription activation domain, and (b) the fusion protein expressed bythe second plasmid comprises a peptide having a DNA binding domain.

In one embodiment of the fusion proteins described herein, the peptidehaving a DNA binding domain is N-terminal relative to the p66 or p51subunit polypeptide. In another embodiment, the peptide having a DNAbinding domain is C-terminal relative to the 51 or p66 subunitpolypeptide. The peptide having a DNA binding domain may be bound in thefusion protein to the p51 or p66 subunit polypeptides. In oneembodiment, they are bound by peptide bonds. Alternatively, the fusionprotein may also comprise one or more additional components, such as apeptide linker and/or an epitope tag. These additional components mayseparate the peptides from the p51 or p66 subunit polypeptide. Thevarious components may be bound to each other by peptide bonds.

In one embodiment of the fusion proteins described herein, the peptidehaving a transcription activation domain is N-terminal relative to thep66 or p51 subunit polypeptide. In another embodiment, the peptidehaving a transcription activation domain is C-terminal relative to the51 or p66 subunit polypeptide. The peptide having a transcriptionactivation domain may be bound in the fusion protein to the p51 or p66subunit polypeptides. In one embodiment, they are bound by peptidebonds. Alternatively, the fusion protein may also comprise one or moreadditional components, such as a peptide linker and/or an epitope tag.These additional components may separate the peptides from the p51 orp66 subunit polypeptide. The various components may be bound to eachother by peptide bonds.

The invention described herein may employ p51 and p66 subunits fromamong various HIV-1 strains. One may use the reverse transcriptasecoding regions for p66 and p51 from any HIV-1 strain. For example, inthe HIV-1 NL4-3 strain, the amino acid and nucleic acid sequences ofwhich may be found in a pNL4-3 clone deposited at Genbank Accession No.M19921. The p51 and p66 subunits share the same N-terminal sequence,whereas p51 does not have the C-terminal ribonuclease H region.Accordingly, p66 corresponds to codons 1-560 and p51 corresponds tocodons 1-440 in the RT gene.

The invention may employ other HIV-1 strains such as the following:HIVE_(HXB2G) (Genbank Accession No. K03455), HIV_(BRUCG) (GenbankAccession No. K02013), HIV_(MNCG) (Genbank Accession No. M17449),HIV_(NY5CG) (Genbank Accession No. M38431); HIV_(JRCSF) (GenbankAccession No.M38429), and HIV_(SF2CG) (Genbank Accession No. K02007).

In one embodiment of the fusion proteins described herein, the DNAbinding domain is a LexA DNA binding domain. The amino acid and nucleicacid sequences for LexA may be found at Genbank Accession No. J01643. Inone embodiment of the methods described herein, the peptide having a DNAbinding domain comprises LexA amino acid residues 1-87. The portion ofLexA which corresponds to amino acid residues 1-87 may comprise a LexADNA binding domain. In one embodiment of the methods described herein,the peptide having a DNA binding domain comprises LexA amino acidresidues 1-202. The portion of LexA which corresponds to amino acidresidues 1-202 may comprise a LexA DNA binding domain.

In one embodiment of the fusion proteins described herein, the DNAbinding domain is a GAL4 DNA binding domain. The amino acid and nucleicacid sequences for Gal4 may be found at Genbank Accession No. K01486.

In one embodiment of the fusion proteins described herein, thetranscription activation domain is a GAL4 transcription activationdomain. In one embodiment, the peptide having a transcription activationdomain comprises GAL4 amino acid residues 768-881. The portion of Gal4which corresponds to amino acid residues 768-881 may comprise a Gal4activation domain.

In one embodiment of the fusion proteins described herein, thetranscription activation domain is a VP16 transcription activationdomain. The amino acid and nucleic acid sequences for VP16 may be foundat Genbank Accession No. U89963.

In one embodiment of the fusion proteins described herein, the fusionprotein expressed by the first plasmid, the second plasmid or bothplasmids comprises a peptide comprising consecutive alanine residues.The above described peptide comprising consecutive alanine residues maybe referred to as an alanine linker. Such linker sequence may be aseries of consecutive amino acid residues other than alanine. Suchlinker sequence may be of various lengths. For example, the linker maycomprise 2 amino acids, 3 amino acids, 4 amino acids, 5 amino acids, 6amino acids, 7 amino acids, 8 amino acids, 9 amino acids or 10 aminoacids. The peptide linker may also be of longer lengths, for example,from about 10 amino acids to about 20 amino acids. In one embodiment,the peptide comprising consecutive alanine residues comprises at least 6alanine residues.

As used herein, the following standard abbreviations are used throughoutthe specification to indicate specific amino acids: A=ala=alanine;R=arg=arginine; N=asn=asparagine; D=asp=aspartic acid; C=cys=cysteine;Q=gln=glutamine; E=glu=glutamic acid; G=gly=glycine; H=his=histidine;I=ile=isoleucine; L=leu=leucine; K=lys=lysine; M=met=methionine;F=phe=phenylalanine; P=pro=proline; S=ser=serine; T=thr=threonine;W=trp=tryptophan; Y=tyr=tyrosine; V=val=valine; B=asx=asparagine oraspartic acid; Z=glx=glutamine or glutamic acid.

As used herein, the following standard abbreviations are used throughoutthe specification to indicate specific nucleotides: C=cytosine;A=adenosine; T=thymidine; G=guanosine; and U=uracil.

In one embodiment of the fusion proteins described herein, the fusionprotein comprises an influenza hemagglutinin (HA) epitope tag. Thesequence for influenza hemagglutinin (HA) epitope may be found inGenbank Accession No. U29899 at nucleotide bases 5042-5068 within theplasmid pACT2. The invention may also comprise other types of epitopetags known to one skilled in the art.

In one embodiment of the fusion proteins described herein, the reportergene is a LacZ reporter gene. The amino acid and nucleic acid sequencesfor LacZ may be found at Genbank Accession no. U89671.

In one embodiment of the methods described herein, (a) the fusionprotein expressed by the first plasmid comprises a peptide comprising aLexA protein DNA binding domain, wherein the p66 subunit polypeptide isbound at its C-terminal amino acid to the N-terminal amino acid of thepeptide comprising a LexA protein DNA binding domain; and (b) the fusionprotein expressed by the second plasmid comprises a Gal4 peptidecorresponding to amino acids 768-881 of Gal4, and an influenzahemagglutinin (HA) epitope tag, which Gal4 peptide is bound at itsC-terminal amino acid to the N-terminal amino acid of the influenzahemagglutinin (HA) epitope tag, which influenza hemagglutinin (HA)epitope tag is bound at its C-terminal amino acid to the N-terminalamino acid of the p51 subunit polypeptide.

In the fusion proteins described herein, the location of the variouscomponents relative to each other may be varied. For example, in theembodiment described above, the peptide comprising a LexA protein DNAbinding domain may alternatively be bound at its C-terminal amino acidto the N-terminal amino acid of the p66 subunit polypeptide. The p51subunit polypeptide may be N-terminal to the Gal4 peptide. One skilledin art would know how to make and use the various vectors and plasmidsdescribed herein.

In one embodiment of the methods described herein, (a) the fusionprotein expressed by the first plasmid comprises a peptide comprising aLexA protein DNA binding domain, wherein the p66 subunit polypeptide isbound at it's C-terminal amino acid to the N-terminal amino acid of thepeptide comprising a LexA protein DNA binding domain; and (b) the fusionprotein expressed by the second plasmid comprises a Gal4 peptidecorresponding to amino acids 768-881 of Gal4, which Gal4 peptide isbound at its C-terminal amino acid to the N-terminal amino acid of thep51 subunit polypeptide.

In one embodiment of the methods described herein, (a) the fusionprotein expressed by the first plasmid comprises a LexA peptidecorresponding to amino acid residues 1-87, wherein the LexA peptide isbound at its C-terminal amino acid to the N-terminal amino acid of theof the p66 subunit polypeptide; and (b) the fusion protein expressed bythe second plasmid comprises a Gal4 peptide corresponding to amino acids768-881 of Gal4, and an influenza hemagglutinin (HA) epitope tag, whichGal4 peptide is bound at its C-terminal amino acid to the N-terminalamino acid of the influenza hemagglutinin (HA) epitope tag, whichinfluenza hemagglutinin (HA) epitope tag is bound at its C-terminalamino acid to the N-terminal amino acid of the p51 subunit polypeptide.

In one embodiment of the methods described herein, (a) the fusionprotein expressed by the first plasmid comprises a LexA peptidecorresponding to amino acid residues 1-87, wherein the LexA peptide isbound at its C-terminal amino acid to the N-terminal amino acid of theof the p66 subunit polypeptide; and (b) the fusion protein expressed bythe second plasmid comprises a Gal4 peptide corresponding to amino acids768-881 of Gal4, which Gal4 peptide is bound at its C-terminal aminoacid to the N-terminal amino acid of the p51 subunit polypeptide.

In one embodiment of the methods described herein, (a) the fusionprotein expressed by the first plasmid comprises a LexA peptidecorresponding to amino acid residues 1-202, and a peptide comprising sixconsecutive alanine residues, wherein the LexA peptide is bound at itsC-terminal amino acid to the N-terminal amino acid of the peptidecomprising six consecutive alanine residues, wherein the peptidecomprising six consecutive alanine residues is bound at its C-terminalamino acid to the N-terminal amino acid of the p66 subunit polypeptide;and (b) the fusion protein expressed by the second plasmid comprises aGal4 peptide corresponding to amino acids 768-881 of Gal4, which Gal4peptide is bound at its C-terminal amino acid to the N-terminal aminoacid of the p51 subunit polypeptide.

In one embodiment of the methods described herein, (a) the fusionprotein expressed by the first plasmid comprises a LexA peptidecorresponding to amino acid residues 1-202, and a peptide comprising sixconsecutive alanine residues, wherein the LexA peptide is bound at itsC-terminal amino acid to the N-terminal amino acid of the peptidecomprising six consecutive alanine residues, wherein the peptidecomprising six consecutive alanine residues is bound at its C-terminalamino acid to the N-terminal amino acid of the p66 subunit polypeptide;and (b) the fusion protein expressed by the second plasmid comprises aGal4 peptide corresponding to amino acids 768-881 of Gal4, and aninfluenza hemagglutinin (HA) epitope tag, which Gal4 peptide is bound atits C-terminal amino acid to the N-terminal amino acid of the influenzahemagglutinin (HA) epitope tag, which influenza hemagglutinin (HA)epitope tag is bound at its C-terminal amino acid to the N-terminalamino acid of the p51 subunit polypeptide.

In one embodiment of the methods described herein, (a) the fusionprotein expressed by the first plasmid comprises a Gal4 peptidecorresponding to amino acids 768-881 of Gal4, an influenza hemagglutinin(HA) epitope tag, and a peptide comprising six consecutive alanineresidues, wherein the Gal4 peptide is bound at its C-terminal amino acidto the N-terminal amino acid of the influenza hemagglutinin (HA) epitopetag, wherein the influenza hemagglutinin (HA) epitope tag is bound atits C-terminal amino acid to the N-terminal amino acid of the peptidecomprising six consecutive alanine residues, wherein the peptidecomprising six consecutive alanine residues is bound at its C-terminalamino acid to the N-terminal amino acid of the p66 subunit polypeptide;and (b) the fusion protein expressed by second plasmid comprises apeptide comprising a LexA protein DNA binding domain, wherein the p51subunit polypeptide is bound at its C-terminal amino acid to theN-terminal amino acid of the peptide comprising a LexA protein DNAbinding domain.

In one embodiment of the methods described herein, (a) the fusionprotein expressed by the first plasmid comprises a Gal4 peptidecorresponding to amino acids 768-881 of Gal4, an influenza hemagglutinin(HA) epitope tag, and a peptide comprising six consecutive alanineresidues, wherein the Gal4 peptide is bound at its C-terminal amino acidto the N-terminal amino acid of the influenza hemagglutinin (HA) epitopetag, wherein the influenza hemagglutinin (HA) epitope tag is bound atits C-terminal amino acid to the N-terminal amino acid of the peptidecomprising six consecutive alanine residues, wherein the peptidecomprising six consecutive alanine residues is bound at its C-terminalamino acid to the N-terminal amino acid of the p66 subunit polypeptide;and (b) the fusion protein expressed by second plasmid comprises apeptide comprising a LexA protein DNA binding domain, wherein peptidecomprising a LexA protein DNA binding domain is bound at its C-terminalamino acid to the N-terminal amino acid of the p51 subunit polypeptide.

In one embodiment of the methods described herein, (a) the fusionprotein expressed by the first plasmid comprises a Gal4 peptidecorresponding to amino acids 768-881 of Gal4, an influenza hemagglutinin(HA) epitope tag, and a peptide comprising six consecutive alanineresidues, wherein the Gal4 peptide is bound at its C-terminal amino acidto the N-terminal amino acid of the influenza hemagglutinin (HA) epitopetag, wherein the influenza hemagglutinin (HA) epitope tag is bound atits C-terminal amino acid to the N-terminal amino acid of the peptidecomprising six consecutive alanine residues, wherein the peptidecomprising six consecutive alanine residues is bound at its C-terminalamino acid to the N-terminal amino acid of the p66 subunit polypeptide;and (b) the fusion protein expressed by second plasmid comprises apeptide comprising a Gal4 protein DNA binding domain, which peptidecomprising a Gal4 protein DNA binding domain is bound at its C-terminalamino acid to the N-terminal amino acid of the p51 subunit polypeptide.

In one embodiment of the methods described herein, (a) the fusionprotein expressed by the first plasmid comprises a Gal4 peptidecorresponding to amino acids 768-881 of Gal4, wherein the Gal4 peptideis bound at its C-terminal amino acid to the N-terminal amino acid ofthe p66 subunit polypeptide; and (b) the fusion protein expressed bysecond plasmid comprises a peptide comprising a LexA protein DNA bindingdomain, wherein the p51 subunit polypeptide is bound at its C-terminalamino acid to the N-terminal amino acid of the peptide comprising a LexAprotein DNA binding domain.

In one embodiment of the methods described herein, (a) the fusionprotein expressed by the first plasmid comprises a Gal4 peptidecorresponding to amino acids 768-881 of Gal4, wherein the Gal4 peptideis bound at its C-terminal amino acid to the N-terminal amino acid ofthe p66 subunit polypeptide; and (b) the fusion protein expressed bysecond plasmid comprises a peptide comprising a LexA protein DNA bindingdomain, which peptide comprising a LexA protein DNA binding domain isbound at its C-terminal amino acid to the N-terminal amino acid of thep51 subunit polypeptide.

In one embodiment of the methods described herein, (a) the fusionprotein expressed by the first plasmid comprises a Gal4 peptidecorresponding to amino acids 768-881 of Gal4, wherein the Gal4 peptideis bound at its C-terminal amino acid to the N-terminal amino acid ofthe p66 subunit polypeptide; and (b) the fusion protein expressed bysecond plasmid comprises a peptide comprising a Gal4 protein DNA bindingdomain, which peptide comprising a Gal4 protein DNA binding domain isbound at its C-terminal amino acid to the N-terminal amino acid of thep51 subunit polypeptide.

In one embodiment of the methods described herein, (a) the fusionprotein expressed by the first plasmid comprises a peptide comprising aLexA protein DNA binding domain, wherein the p66 subunit polypeptide isbound at it's C-terminal amino acid to the N-terminal amino acid of thepeptide comprising a LexA protein DNA binding domain; and (b) the fusionprotein expressed by the second plasmid comprises a Gal4 peptidecorresponding to amino acids 768-881 of Gal4, an influenza hemagglutinin(HA) epitope tag, and a peptide comprising six consecutive alanineresidues, wherein the Gal4 peptide is bound at its C-terminal amino acidto the N-terminal amino acid of the influenza hemagglutinin (HA) epitopetag, wherein the influenza hemagglutinin (HA) epitope tag is bound atits C-terminal amino acid to the N-terminal amino acid of the peptidecomprising six consecutive alanine residues, wherein the peptidecomprising six consecutive alanine residues is bound at its C-terminalamino acid to the N-terminal amino acid of the p66 subunit polypeptide.

This invention provides a method of making a pharmaceutical compositionwhich comprises:

a) determining whether a compound inhibits HIV-1 reverse transcriptaseby one of the methods described herein;

b) recovering the compound if it is determined to inhibit HIV-1 reversetranscriptase; and

c) admixing the compound with a pharmaceutically acceptable carrier.

As used herein, “inhibits” means that the amount is reduced as comparedwith the amount that would occur in a control sample without thecompound. As used herein “enhanced” means that the amount is increasedcompared with the amount that would occur in a control sample withoutthe compound.

As used herein, the term “compound” includes both protein andnon-protein moieties. In one embodiment, the compound is a smallmolecule. In another embodiment, the compound is a protein. The proteinmay be, by way of example, an antibody directed against a portion of ap51 or p66 subunit. The agent may be derived from a library of lowmolecular weight compounds or a library of extracts from plants or otherorganisms. In an embodiment, the agent is known. In a separateembodiment, the agent is not previously known. The agents of the subjectinvention include but are not limited to compounds or molecular entitiessuch as peptides, polypeptides, and other organic or inorganic moleculesand combinations thereof

In one embodiment of the methods described herein, the compound is anantibody or a portion of an antibody. In one embodiment of the antibody,the antibody is a monoclonal antibody. In one embodiment of theantibody, the antibody is a polyclonal antibody. In one embodiment ofthe antibody, the antibody is a humanized antibody. In one embodiment ofthe antibody, the antibody is a chimeric antibody. The portion of theantibody may comprise a light chain of the antibody. The portion of theantibody may comprise a heavy chain of the antibody. The portion of theantibody may comprise a Fab portion of the antibody. The portion of theantibody may comprise a F(ab′)₂ portion of the antibody. The portion ofthe antibody may comprise a Fd portion of the antibody. The portion ofthe antibody may comprise a Fv portion of the antibody. The portion ofthe antibody may comprise a variable domain of the antibody. The portionof the antibody may comprise one or more CDR domains of the antibody.

In one embodiment of the methods described herein, the compound is apolypeptide. In one embodiment of the methods described herein, thecompound is a oligopeptide. In one embodiment of the methods describedherein, the compound is a nonpeptidyl agent. In one embodiment, thenonpeptidyl agent is a compound having a molecular weight less than 500daltons.

In one embodiment of the methods described herein, the reverse HIV-1transcriptase enzyme or its p51 and p66 subunits is present in a subjectand the contacting is effected by administering the compound to thesubject. Accordingly, the subject invention has various applicationswhich includes HIV treatment such as treating a subject who has becomeafflicted with HIV. As used herein, “afflicted with HIV” means that thesubject has at least one cell which has been infected by HIV. As usedherein, “treating” means either slowing, stopping or reversing theprogression of an HIV disorder. In the preferred embodiment, “treating”means reversing the progression to the point of eliminating thedisorder. As used herein, “treating” also means the reduction of thenumber of viral infections, reduction of the number of infectious viralparticles, reduction of the number of virally infected cells, or theamelioration of symptoms associated with HIV. Another application of thesubject invention is to prevent a subject from contracting HIV. As usedherein, “contracting HIV” means becoming infected with HIV, whosegenetic information replicates in and/or incorporates into the hostcells. Another application of the subject invention is to treat asubject who has become infected with HIV. As used herein, “HIVinfection” means the introduction of HIV genetic information into atarget cell, such as by fusion of the target cell membrane with HIV oran HIV envelope glycoprotein⁺ cell. The target cell may be a bodily cellof a subject. In the preferred embodiment, the target cell is a bodilycell from a human subject. Another application of the subject inventionis to inhibit HIV infection. As used herein, “inhibiting HIV infection”means reducing the amount of HIV genetic information introduced into atarget cell population as compared to the amount that would beintroduced without said composition.

This invention provides a method of treating a subject afflicted withHIV which comprises administering to the subject an effective dose of anagent of composition described herein. In one embodiment, the agent orcomposition may be enough to decrease the subject's viral load. As usedherein, “treating” means either slowing, stopping or reversing theprogression of an HIV disorder. In the preferred embodiment, “treating”means reversing the progression to the point of eliminating thedisorder. As used herein, “treating” also means the reduction of thenumber of viral infections, reduction of the number of infectious viralparticles, reduction of the number of virally infected cells, or theamelioration of symptoms associated with HIV. As used herein, “afflictedwith HIV” means that the subject has at least one cell which has beeninfected by HIV.

This invention provides a method of preventing a subject fromcontracting HIV which comprises administering to the subject aneffective dose of an agent or composition described herein.

The dose of the agent or composition of the invention will varydepending on the subject and upon the particular route of administrationused. Dosages can range from 0.1 to 100,000 μg/kg. Based upon thecomposition, the dose can be delivered continuously, such as bycontinuous pump, or at periodic intervals. For example, on one or moreseparate occasions. Desired time intervals of multiple doses of aparticular composition can be determined without undue experimentationby one skilled in the art.

As used herein, “effective dose” means an amount in sufficientquantities to either treat the subject or prevent the subject frombecoming HIV infected. A person of ordinary skill in the art can performsimple titration experiments to determine what amount is required totreat the subject. As used herein, “contracting HIV” means becominginfected with HIV, whose genetic information replicates in and/orincorporates into the host cells. In one embodiment, the effectiveamount of the agent or composition comprises from about 0.000001 mg/kgbody weight to about 100 mg/kg body weight of the subject.

As used herein, “subject” means any animal or artificially modifiedanimal capable of becoming HIV-infected. The subjects include but aremot limited to a human being, a primate, an equine, an opine, an avian,a bovine, a porcine, a canine, a feline or a mouse. Artificiallymodified animals include, but are not limited to, SCID mice with humanimmune systems. The animals include but are not limited to mice, rats,dogs, guinea pigs, ferrets, rabbits, and primates. In the preferredembodiment, the subject is a human being. The subject may be an“HIV-infected subject” which is a subject having at least one of his orher own cells invaded by HIV. In the preferred embodiment, the HIVinfected subject is a human being. The subject may be a“non-HIV-infected subject” which is a subject not having any of his owncells invaded by HIV. In the preferred embodiment, the non-HIV infectedsubject is a human being.

As used herein, “administering” may be effected or performed using anyof the methods known to one skilled in the art, which includesintralesional, intraperitoneal, intramuscular, subcutaneous,intravenous, liposome mediated delivery, transmucosal, intestinal,topical, nasal, oral, anal, ocular or otic delivery.

In one embodiment, amount of the compound administered is between about1 mg and about 50 mg per kg body weight of the subject. In oneembodiment, amount of the compound administered is between about 2 mgand about 40 mg per kg body weight of the subject. In one embodiment,amount of the compound administered is between about 3 mg and about 30mg per kg body weight of the subject. In one embodiment, the amount ofthe compound administered is between about 4 mg and about 20 mg per kgbody weight of the subject. In one embodiment, amount of the compoundadministered is between about 5 mg and about 10 mg per kg body weight ofthe subject.

In one embodiment of the methods described herein, the compound isadministered at least once per day. In one embodiment of the methodsdescribed herein, the agent is administered daily. In one embodiment ofthe methods described herein, the agent is administered every other day.In one embodiment of the methods described herein, the agent isadministered every 6 to 8 days. In one embodiment of the methodsdescribed herein, the agent is administered weekly.

This invention provides a method of inhibiting formation of a complexbetween the p51 subunit polypeptide of HIV-1 reverse transcriptase and ap66 subunit polypeptide of HIV-1 reverse transcriptase, which comprisescontacting either (1) the p51 subunit polypeptide, (2) the p66 subunitpolypeptide, or (3) both the p51 subunit polypeptide and the p66 subunitpolypeptide, with an effective amount of a compound determined to do soby the method of claim 2, so to thereby inhibit formation of a complexbetween the p51 subunit polypeptide of HIV-1 reverse transcriptase and ap66 subunit polypeptide of HIV-1 reverse transcriptase.

This invention provides a method of enhancing formation of a complexbetween the p51 subunit polypeptide of HIV-1 reverse transcriptase and ap66 subunit polypeptide of HIV-1 reverse transcriptase, which comprisescontacting either (1) the p51 subunit polypeptide, (2) the p66 subunitpolypeptide, or (3) both the p51 subunit polypeptide and the p66 subunitpolypeptide, with an effective amount of a compound determined to do soby the method of claim 4, so to thereby enhance formation of a complexbetween the p51 subunit polypeptide of HIV-1 reverse transcriptase and ap66 subunit polypeptide of HIV-1 reverse transcriptase.

This invention provides a method of inhibiting formation of a complexbetween a first p66 subunit polypeptide of HIV-1 reverse transcriptaseand a second p66 subunit polypeptide of HIV-1 reverse transcriptase,which comprises contacting either (1) the first p66 subunit polypeptide,(2) the second p66 subunit polypeptide, or (3) both the first p66subunit polypeptide and the second p66 subunit polypeptide, with aneffective amount of a compound determined to do so by the method ofclaim 6, so to thereby inhibit formation of a complex between the firstp66 subunit polypeptide of HIV-1 reverse transcriptase and the secondp66 subunit polypeptide of HIV-1 reverse transcriptase.

This invention provides a method of enhancing formation of a complexbetween a first p66 subunit polypeptide of HIV-1 reverse transcriptaseand a second p66 subunit polypeptide of HIV-1 reverse transcriptase,which comprises contacting either (1) the first p66 subunit polypeptide,(2) the second p66 subunit polypeptide, or (3) both the first p66subunit polypeptide and the second p66 subunit polypeptide, with aneffective amount of a compound determined to do so by the method ofclaim 8, so to thereby enhance formation of a complex between the firstp66 subunit polypeptide of HIV-1 reverse transcriptase and the secondp66 subunit polypeptide of HIV-1 reverse transcriptase.

In one embodiment of the above methods, the HIV-1 reverse transcriptaseis present in a subject and the contacting is effected by administeringthe compound to the subject. The compound may be administered by variousroutes known to one skilled in the art including but not limited tothose wherein the compound is administered orally, intravenously,subcutaneously, intramuscularly, topically or by liposome-mediateddelivery. The subject may be any subject including but not limited to ahuman being, a primate, an equine, an opine, an avian, a bovine, aporcine, a canine, a feline or a mouse. In one embodiment, the compoundis administered at least once per day. In one embodiment, the compoundis administered daily. In one embodiment, the compound is administeredevery other day. In one embodiment, compound is administered every 6 to8 days. In one embodiment, the compound is administered weekly.

This invention provides a compound determined to be capable ofinhibiting formation of a complex between a p51 subunit polypeptide ofHIV-1 reverse transcriptase and a p66 subunit polypeptide of HIV-1reverse transcriptase.

This invention provides a compound determined to be capable of enhancingformation of a complex between a p51 subunit polypeptide of HIV-1reverse transcriptase and a p66 subunit polypeptide of HIV-1 reversetranscriptase.

This invention provides a compound determined to be capable ofinhibiting formation of a complex between a first p66 subunitpolypeptide of HIV-1 reverse transcriptase and a second p66 subunitpolypeptide of HIV-1 reverse transcriptase.

This invention provides a compound determined to be capable of enhancingformation of a complex between a first p66 subunit polypeptide of HIV-1reverse transcriptase and a second p66 subunit polypeptide of HIV-1reverse transcriptase.

This invention provides a composition which comprises one of thecompounds described herein and a carrier. As used herein, “composition”means a mixture. The compositions include but are not limited to thosesuitable for oral, rectal, intravaginal, topical, nasal, opthalmic, orparenteral, intravenous, subcutaneous, intramuscular, andintraperitoneal administration to a subject. As used herein,“parenteral” includes but is not limited to subcutaneous, intravenous,intramuscular, or intrasternal injections or infusion techniques.

This invention provides an agent or composition described herein and acarrier. Such carrier may be one that is a pharmaceutically acceptablecarrier. Pharmaceutically acceptable carriers are well known to thoseskilled in the art. Such pharmaceutically acceptable carriers mayinclude but are not limited to aqueous or non-aqueous solutions,suspensions, and emulsions. Examples of non-aqueous solvents arepropylene glycol, polyethylene glycol, vegetable oils such as olive oil,and injectable organic esters such as ethyl oleate. Aqueous carriersinclude water, alcoholic/aqueous solutions, emulsions or suspensions,saline and buffered media. Parenteral vehicles include sodium chloridesolution, Ringer's dextrose, dextrose and sodium chloride, lactatedRinger's or fixed oils. Intravenous vehicles include fluid and nutrientreplenishers, electrolyte replenishers such as those based on Ringer'sdextrose, and the like. Preservatives and other additives may also bepresent, such as, for example, antimicrobials, antioxidants, chelatingagents, inert gases and the like.

In one embodiment of the agents described herein, the compound is anantibody or portion of an antibody. As used herein, “antibody” means animmunoglobulin molecule comprising two heavy chains and two light chainsand which recognizes an antigen. The immunoglobulin molecule may derivefrom any of the commonly known classes, including but not limited toIgA, secretory IgA, IgG and IgM. IgG subclasses are also well known tothose in the art and include but are not limited to human IgG1, IgG2,IgG3 and IgG4. It includes, by way of example, both naturally occurringand non-naturally occurring antibodies. Specifically, “antibody”includes polyclonal and monoclonal antibodies, and monovalent anddivalent fragments thereof. Furthermore, “antibody” includes chimericantibodies, wholly synthetic antibodies, single chain antibodies, andfragments thereof. optionally, an antibody can be labeled with adetectable marker. Detectable markers include, for example, radioactiveor fluorescent markers. The antibody may be a human or nonhumanantibody. The nonhuman antibody may be humanized by recombinant methodsto reduce its immunogenicity in man. Methods for humanizing antibodiesare known to those skilled in the art. As used herein, “monoclonalantibody,” also designated as mAb, is used to describe antibodymolecules whose primary sequences are essentially identical and whichexhibit the same antigenic specificity. Monoclonal antibodies may beproduced by hybridoma, recombinant, transgenic or other techniques knownto one skilled in the art. The term “antibody” includes, but is notlimited to, both naturally occurring and non-naturally occurringantibodies. Specifically, the term “antibody” includes polyclonal andmonoclonal antibodies, and antigen-binding fragments thereof.Furthermore, the term “antibody” includes chimeric antibodies, whollysynthetic antibodies, and antigen-binding fragments thereof.Accordingly, in one embodiment, the antibody is a monoclonal antibody.In one embodiment, the antibody is a polyclonal antibody. In oneembodiment, the antibody is a humanized antibody. In one embodiment, theantibody is a chimeric antibody. Such chimeric antibodies may comprise aportion of an antibody from one source and a portion of an antibody fromanother source.

In one embodiment, the portion of the antibody comprises a light chainof the antibody. As used herein, “light chain” means the smallerpolypeptide of an antibody molecule composed of one variable domain (VL)and one constant domain (CL), or fragments thereof. In one embodiment,the portion of the antibody comprises a heavy chain of the antibody. Asused herein, “heavy chain” means the larger polypeptide of an antibodymolecule composed of one variable domain (VH) and three or four constantdomains (CH1, CH2, CH3, and CH4), or fragments thereof. In oneembodiment, the portion of the antibody comprises a Fab portion of theantibody. As used herein, “Fab” means a monovalent antigen bindingfragment of an immunoglobulin that consists of one light chain and partof a heavy chain. It can be obtained by brief papain digestion or byrecombinant methods. In one embodiment, the portion of the antibodycomprises a F(ab′)₂ portion of the antibody. As used herein, “F(ab′)2fragment” means a bivalent antigen binding fragment of an immunoglobulinthat consists of both light chains and part of both heavy chains. It canbe obtained by brief pepsin digestion or recombinant methods. In oneembodiment, the portion of the antibody comprises a Fd portion of theantibody. In one embodiment, the portion of the antibody comprises a Fvportion of the antibody. In one embodiment, the portion of the antibodycomprises a variable domain of the antibody. In one embodiment, theportion of the antibody comprises a constant domain of the antibody. Inone embodiment, the portion of the antibody comprises one or more CDRdomains of the antibody. As used herein, “CDR” or “complementaritydetermining region” means a highly variable sequence of amino acids inthe variable domain of an antibody.

This invention provides humanized forms of the antibodies describedherein. As used herein, “humanized” describes antibodies wherein some,most or all of the amino acids outside the CDR regions are replaced withcorresponding amino acids derived from human immunoglobulin molecules.In one embodiment of the humanized forms of the antibodies, some, mostor all of the amino acids outside the CDR regions have been replacedwith amino acids from human immunoglobulin molecules but where some,most or all amino acids within one or more CDR regions are unchanged.Small additions, deletions, insertions, substitutions or modificationsof amino acids are permissible as long as they would not abrogate theability of the antibody to bind a given antigen. Suitable humanimmunoglobulin molecules would include IgG1, IgG2, IgG3, IgG4, IgA andIgM molecules. A “humanized” antibody would retain a similar antigenicspecificity as the original antibody.

One skilled in the art would know how to make the humanized antibodiesof the subject invention. Various publications, several of which arehereby incorporated by reference into this application, also describehow to make humanized antibodies. For example, the methods described inU.S. Pat. No. 4,816,567 comprise the production of chimeric antibodieshaving a variable region of one antibody and a constant region ofanother antibody.

U.S. Pat. No. 5,225,539 describes another approach for the production ofa humanized antibody. This patent describes the use of recombinant DNAtechnology to produce a humanized antibody wherein the CDRs of avariable region of one immunoglobulin are replaced with the CDRs from animmunoglobulin with a different specificity such that the humanizedantibody would recognize the desired target but would not be recognizedin a significant way by the human subject's immune system. Specifically,site directed mutagenesis is used to graft the CDRs onto the framework.

Other approaches for humanizing an antibody are described in U.S. Pat.Nos. 5,585,089 and 5,693,761 and WO 90/07861 which describe methods forproducing humanized immunoglobulins. These have one or more CDRs andpossible additional amino acids from a donor immunoglobulin and aframework region from an accepting human immunoglobulin. These patentsdescribe a method to increase the affinity of an antibody for thedesired antigen. Some amino acids in the framework are chosen to be thesame as the amino acids at those positions in the donor rather than inthe acceptor. Specifically, these patents describe the preparation of ahumanized antibody that binds to a receptor by combining the CDRs of amouse monoclonal antibody with human immunoglobulin framework andconstant regions. Human framework regions can be chosen to maximizehomology with the mouse sequence. A computer model can be used toidentify amino acids in the framework region which are likely tointeract with the CDRs or the specific antigen and then mouse aminoacids can be used at these positions to create the humanized antibody.

The above patents U.S. Pat. Nos. 5,585,089 and 5,693,761, and WO90/07861 also propose four possible criteria which may used in designingthe humanized antibodies. The first proposal was that for an acceptor,use a framework from a particular human immunoglobulin that is unusuallyhomologous to the donor immunoglobulin to be humanized, or use aconsensus framework from many human antibodies. The second proposal wasthat if an amino acid in the framework of the human immunoglobulin isunusual and the donor amino acid at that position is typical for humansequences, then the donor amino acid rather than the acceptor may beselected. The third proposal was that in the positions immediatelyadjacent to the 3 CDRs in the humanized immunoglobulin chain, the donoramino acid rather than the acceptor amino acid may be selected. Thefourth proposal was to use the donor amino acid reside at the frameworkpositions at which the amino acid is predicted to have a side chain atomwithin 3Å of the CDRs in a three dimensional model of the antibody andis predicted to be capable of interacting with the CDRs. The abovemethods are merely illustrative of some of the methods that one skilledin the art could employ to make humanized antibodies.

This invention provides isolated nucleic acids encoding the antibodiesdescribed herein or their humanized versions. The nucleic acid can beRNA, DNA or cDNA. In one embodiment, the nucleic acid encodes the lightchain. In one embodiment, the nucleic acid encodes the heavy chain. Inone embodiment, the nucleic acid encodes both the heavy and lightchains. In one embodiment, one or more nucleic acids encode the Fabportion.

In one embodiment, one or more nucleic acids encode CDR portions. In oneembodiment, the nucleic acid encodes the variable domain. In oneembodiment of the agents described herein, the agent is a polypeptide.In one embodiment of the agents described herein, the agent is aoligopeptide. As used herein, “polypeptide” means two or more aminoacids linked by a peptide bond.

The nucleic acids, polyepeptides and antibodies described herein may beisolated and/or purified. One skilled in the art would know how toisolate and/or purify them. Methods are provided in any laboratorymanual such as “Molecular Cloning” by Samrook, Fritsch and Maniatis.

In one embodiment of the agents described herein, the compound is anonpeptidyl agent. As used herein, “nonpeptidyl agent” means an agentthat does not consist in its entirety of a linear sequence of aminoacids linked by peptide bonds. A nonpeptidyl molecule may, however,contain one or more peptide bonds. In one embodiment, the nonpeptidylagent is a compound having a molecular weight less than 500 daltons. Asused herein, a “small molecule” is one having a molecular weight lessthan 500 daltons.

The polypeptides described herein may be made by any means known to oneskilled in the art. For example, the protein may be made by recombinantexpression from a nucleic acid, such as a plasmid or vector comprisingthe encoding nucleic acid, wherein the plasmid or vector is in asuitable host cell, i.e. a host-vector system for the production of thepolypeptide of interest. A suitable vector may be made which comprisessuitable regulatory sequences, such as enhancers and promoters. The hostcell may be of any type, including but not limited to mammalian,bacteria and yeast cells. Suitable bacterial cells include E. colicells. Suitable mammalian cells include but are not limited to humanembryonic kidney (HEK) 293T cells, HeLa cells, NIH 3T3 cells Chinesehamster ovary (CHO) cells and Cos cells.

This invention provides a method of testing a compound to determinewhether it is an inhibitor of formation of a complex between a p66subunit polypeptide of HIV-1 reverse transcriptase and a p51 subunitpolypeptide of HIV-1 reverse transcriptase which comprises: a)contacting a yeast cell cotransformed with a first plasmid whichexpresses in the cell a fusion protein comprising the p66 subunitpolypeptide of HIV-1 reverse transcriptase and a second plasmid whichexpresses in the cell a fusion protein comprising the p51 subunitpolypeptide of HIV-1 reverse transcriptase with the compound wherein thecell further comprises a reporter gene which is activated in thepresence of a complex between the p66 subunit polypeptide and the p51subunit polypeptide; b) determining the level of activity of thereporter gene in the cell in the presence of the compound; and c)comparing the level of activity of the reporter gene determined in step(b) with the level of activity of the reporter gene in the absence ofthe compound, wherein a decreased level of activity of the reporter geneindicates that the compound is an inhibitor of the formation of thecomplex between the p51 subunit polypeptide of HIV-1 reversetranscriptase and the p66 subunit polypeptide of HIV-1 reversetranscriptase.

In an embodiment of the above-described method of testing a compound todetermine whether it is an inhibitor of formation of a complex between ap66 subunit polypeptide of HIV-1 reverse transcriptase and a p51 subunitpolypeptide of HIV-1 reverse transcriptase, the fusion protein expressedby the first plasmid further comprises full length bacterial proteinLexA fused to the p66 subunit polypeptide of HIV-1 reverse transcriptaseat amino acid position 1 of the N-terminal amino acid sequence of thefull length bacterial protein LexA and the fusion protein expressed bythe second plasmid further comprises amino acids at positions 768-881 ofthe C-terminal amino acid sequence of Gal4AD, wherein the C-terminalamino acid sequence of Gal4AD is fused at amino acid position 881 to oneend of an influenza hemagglutinin (HA) epitope tag, wherein the p51subunit polypeptide is fused to the second end of the influenza HAepitope tag.

In another embodiment of the above-described method of testing acompound to determine whether it is an inhibitor of formation of acomplex between a p66 subunit polypeptide of HIV-1 reverse transcriptaseand a p51 subunit polypeptide of HIV-1 reverse transcriptase, the fusionprotein expressed by the first plasmid further comprises full lengthbacterial protein LexA fused to the p66 subunit polypeptide of HIV-1reverse transcriptase at amino acid position 1 of the N-terminal aminoacid sequence of the full length bacterial protein LexA and the fusionprotein expressed by the second plasmid further comprises amino acids atpositions 768-881 of the C-terminal amino acid sequence of Gal4AD fusedat position 881 to the p51 subunit polypeptide of HIV-1 reversetranscriptase.

In still another embodiment of the above-described method of testing acompound to determine whether it is an inhibitor of formation of acomplex between a p66 subunit polypeptide of HIV-1 reverse transcriptaseand a p51 subunit polypeptide of HIV-1 reverse transcriptase, the fusionprotein expressed by the first plasmid further comprises amino acids1-87 of the LexA DNA binding domain fused to the p66 subunit polypeptideof HIV-1 reverse transcriptase at amino acid position 87 and the fusionprotein expressed by the second plasmid further comprises amino acids atpositions 768-881 of the C-terminal amino acid sequence of Gal4AD,wherein the C-terminal amino acid sequence of Gal4AD is fused at aminoacid position 881 to one end of an influenza hemagglutinin (HA) epitopetag, wherein the p51 subunit polypeptide is fused to the second end ofthe influenza HA epitope tag.

In a further embodiment of the above-described method of testing acompound to determine whether it is an inhibitor of formation of acomplex between a p66 subunit polypeptide of HIV-1 reverse transcriptaseand a p51 subunit polypeptide of HIV-1 reverse transcriptase, the fusionprotein expressed by the first plasmid further comprises amino acids1-87 of the LexA DNA binding domain fused to the p66 subunit polypeptideof HIV-1 reverse transcriptase at amino acid position 87 and the fusionprotein expressed by the second plasmid further comprises amino acids atpositions 768-881 of the C-terminal amino acid sequence of Gal4AD fusedat position 881 to the p51 subunit polypeptide of HIV-1 reversetranscriptase.

In a still further embodiment of the above-described method of testing acompound to determine whether it is an inhibitor of formation of acomplex between a p66 subunit polypeptide of HIV-1 reverse transcriptaseand a p51 subunit polypeptide of HIV-1 reverse transcriptase, the fusionprotein expressed by the first plasmid further comprises full lengthbacterial protein LexA protein fused at amino acid position 202 to afirst end of a six alanine linker, wherein the p66 subunit polypeptideis fused at amino acid 1 to the second end of the six alanine linker andthe fusion protein expressed by the second plasmid expresses furthercomprises amino acids at positions 768-881 of the C-terminal amino acidsequence of Gal4AD fused at position 881 to the p51 subunit polypeptideof HIV-1 reverse transcriptase.

In yet another embodiment of the above-described method of testing acompound to determine whether it is an inhibitor of formation of acomplex between a p66 subunit polypeptide of HIV-1 reverse transcriptaseand a p51 subunit polypeptide of HIV-1 reverse transcriptase, the fusionprotein expressed by the first plasmid further comprises full lengthbacterial protein LexA protein fused at amino acid position 202 to oneend of a six alanine linker and the p66 subunit polypeptide fused atamino acid 1 to the other end of the six alanine linker and the fusionprotein expressed by the second plasmid further comprises amino acids atpositions 768-881 of the C-terminal amino acid sequence of Gal4ADwherein said C-terminal sequence is fused at amino acid position 881 toone end of an influenza hemagglutinin (HA) epitope tag and the p51subunit polypeptide is fused at the other end of the HA epitope tag.

In another embodiment of the above-described method of testing acompound to determine whether it is an inhibitor of formation of acomplex between a p66 subunit polypeptide of HIV-1 reverse transcriptaseand a p51 subunit polypeptide of HIV-1 reverse transcriptase, the fusionprotein expressed by the first plasmid further comprises amino acids atpositions 768-881 of the C-terminal amino acid sequence of Gal4AD fusedto a first end of an influenza hemagglutinin (HA) epitope and a sixalanine linker fused at a first end to the second end of the influenzahemagglutinin (HA) epitope, wherein the p66 subunit polypeptide of HIV-1reverse transcriptase is fused at amino acid 1 to the second end of thesix alanine linker and the fusion protein expressed by second plasmidfurther comprises full length LexA bacterial protein LexA fused at aminoacid 1 to the p51 subunit polypeptide of HIV-1 reverse transcriptase.

In a further embodiment of the above-described method of testing acompound to determine whether it is an inhibitor of formation of acomplex between a p66 subunit polypeptide of HIV-1 reverse transcriptaseand a p51 subunit polypeptide of HIV-1 reverse transcriptase, the fusionprotein expressed by the first plasmid further comprises amino acids atpositions 768-881 of the C-terminal amino acid sequence of Gal4AD fusedto a first end of an influenza hemagglutinin (HA) epitope and a sixalanine linker fused at a first end to the second end of the influenzahemagglutinin (HA) epitope, wherein the p66 subunit polypeptide of HIV-1reverse transcriptase is fused at amino acid 1 to the second end of thesix alanine linker and the fusion protein expressed by the secondplasmid further comprises the LexA DNA binding domain fused to aminoacid position 1 of the p51 subunit polypeptide of HIV-1 reversetranscriptase.

In a further embodiment of the above-described method of testing acompound to determine whether it is an inhibitor of formation of acomplex between a p66 subunit polypeptide of HIV-1 reverse transcriptaseand a p51 subunit polypeptide of HIV-1 reverse transcriptase, the fusionprotein expressed by the first plasmid further comprises amino acids atpositions 768-881 of the C-terminal amino acid sequence of Gal4AD fusedto a first end of an influenza hemagglutinin (HA) epitope and a sixalanine linker fused at a first end to the second end of the influenzahemagglutinin (HA) epitope, wherein the p66 subunit polypeptide of HIV-1reverse transcriptase is fused at amino acid 1 to the second end of thesix alanine linker and the fusion protein expressed by the secondplasmid further comprises the GAL4 DNA binding domain fused to aminoacid position 1 of the p51 subunit polypeptide of HIV-1 reversetranscriptase.

In still another embodiment of the above-described method of testing acompound to determine whether it is an inhibitor of formation of acomplex between a p66 subunit polypeptide of HIV-1 reverse transcriptaseand a p51 subunit polypeptide of HIV-1 reverse transcriptase, the fusionprotein expressed by the first plasmid further comprises amino acids atpositions 768-881 of the C-terminal amino acid sequence of Gal4AD,wherein the C-terminal amino acid sequence of Gal4AD is fused at aminoacid position 881 to the p66 subunit polypeptide of HIV-1 reversetranscriptase, and the fusion protein expressed by second plasmidfurther comprises full length LexA bacterial protein LexA fused at aminoacid position 1 to the p51 subunit polypeptide of HIV-1 reversetranscriptase.

In another embodiment of the above-described method of testing acompound to determine whether it is an inhibitor of formation of acomplex between a p66 subunit polypeptide of HIV-1 reverse transcriptaseand a p51 subunit polypeptide of HIV-1 reverse transcriptase, the fusionprotein expressed by the first plasmid further comprises amino acids atpositions 768-881 of the C-terminal amino acid sequence of Gal4AD fusedat amino acid position 881 to the p66 subunit polypeptide of HIV-1reverse transcriptase and the fusion protein expressed by the secondplasmid further comprises the LexA DNA binding domain fused to aminoacid position 1 of the p51 subunit polypeptide of HIV-1 reversetranscriptase.

In a further embodiment of the above-described method of testing acompound to determine whether it is an inhibitor of formation of acomplex between a p66 subunit polypeptide of HIV-1 reverse transcriptaseand a p51 subunit polypeptide of HIV-1 reverse transcriptase, the fusionprotein expressed by the first plasmid further comprises amino acids atpositions 768-881 of the C-terminal amino acid sequence of Gal4AD fusedat amino acid position 881 to the p66 subunit polypeptide of HIV-1reverse transcriptase and the fusion protein expressed by the secondplasmid further comprises the GAL4 DNA binding domain fused to aminoacid position 1 of the p51 subunit polypeptide of HIV-1 reversetranscriptase.

One of skill will readily be able to make or use the plasmids describedherein using the known nucleic acid sequence for HIV-1 reversetranscriptase, and the p66 subunit polypeptide thereof or the p51subunit polypeptide thereof which may be comprised in expression vectorsmade by one of ordinary skill in the art; and make or purchase thevectors used herein comprising the full length LexA protein or truncatedportions thereof, i.e. the lexA DNA binding domain, the GAL4 DNA bindingdomain, and GAL4 activation domain (GAL4AD) and with an HA epitope tagbetween the (GAL4AD) and the polylinker.

The inhibitors determined by the above-described methods are useful forthe preparation of drugs, as pharmaceutical compositions, which willblock complex formation between the p66 subunit polypeptide of HIV-1reverse transcriptase and the p51 subunit polypeptide of HIV-1 reversetranscriptase so as to kill the HIV-1 virus or render it inactive orinacabable of infecting cells of a subject, including a human subject.

This invention also provides a method of making a pharmaceuticalcomposition comprising an inhibitor of the formation of the complexbetween the p66 subunit polypeptide of HIV-1 reverse transcriptase andthe p51 subunit polypeptide of HIV-1 reverse transcriptase whichcomprises:

a) determining whether a compound is an inhibitor of the formation ofthe complex between the p66 subunit polypeptide of HIV-1 reversetranscriptase and the p51 subunit polypeptide of HIV-1 reversetranscriptase according to a method which comprises: i) contacting ayeast cell cotransformed with a first plasmid which expresses in thecell a fusion protein comprising the p66 subunit polypeptide of HIV-1reverse transcriptase and a second plasmid which expresses in the cell afusion protein comprising the p51 subunit polypeptide of HIV-1 reversetranscriptase with the compound wherein the cell further comprises areporter gene which is activated in the presence of a complex betweenthe p66 subunit polypeptide and the p51 subunit polypeptide; ii)determining the level of activity of the reporter gene in the cell inthe presence of the compound; and iii) comparing the level of activityof the reporter gene determined in step (ii) with the level of activityof the reporter gene in the absence of the compound, wherein a decreasedlevel of activity of the reporter gene indicates that the compound is aninhibitor of the formation of the complex between the p51 subunitpolypeptide of HIV-1 reverse transcriptase and the p66 subunitpolypeptide of HIV-1 reverse transcriptase; and

b) admixing the compound determined to be the inhibitor in step (a)(iii)with a pharmaceutically acceptable carrier. Any of the above-describedmethods to determine whether a compound is an inhibitor of the formationof the complex between the p66 subunit polypeptide of HIV-1 reversetranscriptase and the p51 subunit polypeptide of HIV-1 reversetranscriptase may be used in the method of making a pharmaceuticalcomposition comprising the determined inhibitor compound, but is notlimited thereto, since one of skill will readily be able to substitutewell known reporter genes for the reporter genes used in the examplesherein. Moreover, one of skill is not limited to using the yeast cellsexemplified in any of the above-described methods herein, but may modifythe methods to use other eukaryotic cells, mammalian cells or cell linessuch as 298 T cells.

This invention further provides a method of testing a compound todetermine whether it is an activator of formation of a complex between ap66 subunit polypeptide of HIV-1 reverse transcriptase and a p51 subunitpolypeptide of HIV-1 reverse transcriptase which comprises: a)contacting a yeast cell cotransformed with a first plasmid whichexpresses in the cell a fusion protein comprising the p66 subunitpolypeptide of HIV-1 reverse transcriptase and a second plasmid whichexpresses in the cell a fusion protein comprising the p51 subunitpolypeptide of HIV-1 reverse transcriptase with the compound wherein thecell further comprises a reporter gene which is activated in thepresence of a complex between the p66 subunit polypeptide and the p51subunit polypeptide; b) determining the level of activity of thereporter gene in the cell in the presence of the compound; and c)comparing the level of activity of the reporter gene determined in step(b) with the level of activity of the reporter gene in the absence ofthe compound, wherein an increased level of activity of the reportergene indicates that the compound is an activator of the formation of thecomplex between the p51 subunit polypeptide of HIV-1 reversetranscriptase and the p66 subunit polypeptide of HIV-1 reversetranscriptase.

In an embodiment of the above-described method of testing a compound todetermine whether it is an activator of formation of a complex between ap66 subunit polypeptide of HIV-1 reverse transcriptase and a p51 subunitpolypeptide of HIV-1 reverse transcriptase, the fusion protein expressedby the first plasmid further comprises full length bacterial proteinLexA fused to the p66 subunit polypeptide of HIV-1 reverse transcriptaseat amino acid position 1 of the N-terminal amino acid sequence of thefull length bacterial protein LexA and the fusion protein expressed bythe second plasmid further comprises amino acids at positions 768-881 ofthe C-terminal amino acid sequence of Gal4AD, wherein the C-terminalamino acid sequence of Gal4AD is fused at amino acid position 881 to oneend of an influenza hemagglutinin (HA) epitope tag, wherein the p51subunit polypeptide is fused to the second end of the influenza HAepitope tag.

In another embodiment of the above-described method of testing acompound to determine whether it is an activator of formation of acomplex between a p66 subunit polypeptide of HIV-1 reverse transcriptaseand a p51 subunit polypeptide of HIV-1 reverse transcriptase, the fusionprotein expressed by the first plasmid further comprises full lengthbacterial protein LexA fused to the p66 subunit polypeptide of HIV-1reverse transcriptase at amino acid position 1 of the N-terminal aminoacid sequence of the full length bacterial protein LexA and the fusionprotein expressed by the second plasmid further comprises amino acids atpositions 768-881 of the C-terminal amino acid sequence of Gal4AD fusedat position 881 to the p51 subunit polypeptide of HIV-1 reversetranscriptase.

In a further embodiment of the above-described method of testing acompound to determine whether it is an activator of formation of acomplex between a p66 subunit polypeptide of HIV-1 reverse transcriptaseand a p51 subunit polypeptide of HIV-1 reverse transcriptase, the fusionprotein expressed by the first plasmid further comprises amino acids1-87 of the LexA DNA binding domain fused to the p66 subunit polypeptideof HIV-1 reverse transcriptase at amino acid position 87 and the fusionprotein expressed by the second plasmid further comprises amino acids atpositions 768-881 of the C-terminal amino acid sequence of Gal4AD,wherein the C-terminal amino acid sequence of Gal4AD is fused at aminoacid position 881 to one end of an influenza hemagglutinin (HA) epitopetag, wherein the p51 subunit polypeptide is fused to the second end ofthe influenza HA epitope tag.

In another embodiment of the above-described method of testing acompound to determine whether it is an activator of formation of acomplex between a p66 subunit polypeptide of HIV-1 reverse transcriptaseand a p51 subunit polypeptide of HIV-1 reverse transcriptase, the fusionprotein expressed by the first plasmid further comprises amino acids1-87 of the LexA DNA binding domain fused to the p66 subunit polypeptideof HIV-1 reverse transcriptase at amino acid position 87 and the fusionprotein expressed by the second plasmid further comprises amino acids atpositions 768-881 of the C-terminal amino acid sequence of Gal4AD fusedat position 881 to the p51 subunit polypeptide of HIV-1 reversetranscriptase.

In a still further embodiment of the above-described method of testing acompound to determine whether it is an activator of formation of acomplex between a p66 subunit polypeptide of HIV-1 reverse transcriptaseand a p51 subunit polypeptide of HIV-1 reverse transcriptase, the fusionprotein expressed by the first plasmid further comprises full lengthbacterial protein LexA protein fused at amino acid position 202 to afirst end of a six alanine linker, wherein the p66 subunit polypeptideis fused at amino acid 1 to the second end of the six alanine linker andthe fusion protein expressed by the second plasmid expresses furthercomprises amino acids at positions 768-881 of the C-terminal amino acidsequence of Gal4AD fused at position 881 to the p51 subunit polypeptideof HIV-1 reverse transcriptase.

In another embodiment of the above-described method of testing acompound to determine whether it is an activator of formation of acomplex between a p66 subunit polypeptide of HIV-1 reverse transcriptaseand a p51 subunit polypeptide of HIV-1 reverse transcriptase, the fusionprotein expressed by the first plasmid further comprises full lengthbacterial protein LexA protein fused at amino acid position 202 to oneend of a six alanine linker and the p66 subunit polypeptide fused atamino acid 1 to the other end of the six alanine linker and the fusionprotein expressed by the second plasmid further comprises amino acids atpositions 768-881 of the C-terminal amino acid sequence of Gal4ADwherein said C-terminal sequence is fused at amino acid position 881 toone end of an influenza hemagglutinin (HA) epitope tag and the p51subunit polypeptide is fused at the other end of the HA epitope tag.

In a further embodiment of the above-described method of testing acompound to determine whether it is an activator of formation of acomplex between a p66 subunit polypeptide of HIV-1 reverse transcriptaseand a p51 subunit polypeptide of HIV-1 reverse transcriptase, the fusionprotein expressed by the first plasmid further comprises amino acids atpositions 768-881 of the C-terminal amino acid sequence of Gal4AD fusedto a first end of an influenza hemagglutinin (HA) epitope and a sixalanine linker fused at a first end to the second end of the influenzahemagglutinin (HA) epitope, wherein the p66 subunit polypeptide of HIV-1reverse transcriptase is fused at amino acid 1 to the second end of thesix alanine linker and the fusion protein expressed by second plasmidfurther comprises full length LexA bacterial protein LexA fused at aminoacid 1 to the p51 subunit polypeptide of HIV-1 reverse transcriptase.

In another embodiment of the above-described method of testing acompound to determine whether it is an activator of formation of acomplex between a p66 subunit polypeptide of HIV-1 reverse transcriptaseand a p51 subunit polypeptide of HIV-1 reverse transcriptase, the fusionprotein expressed by the first plasmid further comprises amino acids atpositions 768-881 of the C-terminal amino acid sequence of Gal4AD fusedto a first end of an influenza hemagglutinin (HA) epitope and a sixalanine linker fused at a first end to the second end of the influenzahemagglutinin (HA) epitope, wherein the p66 subunit polypeptide of HIV-1reverse transcriptase is fused at amino acid 1 to the second end of thesix alanine linker and the fusion protein expressed by the secondplasmid further comprises the LexA DNA binding domain fused to aminoacid position 1 of the p51 subunit polypeptide of HIV-1 reversetranscriptase.

In still another embodiment of the above-described method of testing acompound to determine whether it is an activator of formation of acomplex between a p66 subunit polypeptide of HIV-1 reverse transcriptaseand a p51 subunit polypeptide of HIV-1 reverse transcriptase, the fusionprotein expressed by the first plasmid further comprises amino acids atpositions 768-881 of the C-terminal amino acid sequence of Gal4AD fusedto a first end of an influenza hemagglutinin (HA) epitope and a sixalanine linker fused at a first end to the second end of the influenzahemagglutinin (HA) epitope, wherein the p66 subunit polypeptide of HIV-1reverse transcriptase is fused at amino acid 1 to the second end of thesix alanine linker and the fusion protein expressed by the secondplasmid further comprises the GAL4 DNA binding domain fused to aminoacid position 1 of the p51 subunit polypeptide of HIV-1 reversetranscriptase.

In a further embodiment of the above-described method of testing acompound to determine whether it is an activator of formation of acomplex between a p66 subunit polypeptide of HIV-1 reverse transcriptaseand a p51 subunit polypeptide of HIV-1 reverse transcriptase, the fusionprotein expressed by the first plasmid further comprises amino acids atpositions 768-881 of the C-terminal amino acid sequence of Gal4AD,wherein the C-terminal amino acid sequence of Gal4AD is fused at aminoacid position 881 to the p66 subunit polypeptide of HIV-1 reversetranscriptase, and the fusion protein expressed by second plasmidfurther comprises full length LexA bacterial protein LexA fused at aminoacid position 1 to the p51 subunit polypeptide of HIV-1 reversetranscriptase.

In another embodiment of the above-described method of testing acompound to determine whether it is an activator of formation of acomplex between a p66 subunit polypeptide of HIV-1 reverse transcriptaseand a p51 subunit polypeptide of HIV-1 reverse transcriptase, the fusionprotein expressed by the first plasmid further comprises amino acids atpositions 768-881 of the C-terminal amino acid sequence of Gal4AD fusedat amino acid position 881 to the p66 subunit polypeptide of HIV-1reverse transcriptase and the fusion protein expressed by the secondplasmid further comprises the LexA DNA binding domain fused to aminoacid position 1 of the p51 subunit polypeptide of HIV-1 reversetranscriptase.

In a further embodiment of the above-described method of testing acompound to determine whether it is an activator of formation of acomplex between a p66 subunit polypeptide of HIV-1 reverse transcriptaseand a p51 subunit polypeptide of HIV-1 reverse transcriptase, the fusionprotein expressed by the first plasmid further comprises amino acids atpositions 768-881 of the C-terminal amino acid sequence of Gal4AD fusedat amino acid position 881 to the p66 subunit polypeptide of HIV-1reverse transcriptase and the fusion protein expressed by the secondplasmid further comprises the GAL4 DNA binding domain fused to aminoacid position 1 of the p51 subunit polypeptide of HIV-1 reversetranscriptase.

The activators determined by the above-described methods are useful forthe preparation of drugs, as pharmaceutical compositions, which willenhance complex formation prematurely or inappropriately between the p66subunit polypeptide of HIV-1 reverse transcriptase and the p51 subunitpolypeptide of HIV-1 reverse transcriptase so as to kill the HIV-1 virusor render the the HIV-1 virus inactive or incapable of infecting cellsof a subject, i.e. lack the functions of an infected HIV-1 virus,including human subjects.

This invention also provides a method of making a pharmaceuticalcomposition comprising an activator of the formation of the complexbetween the p66 subunit polypeptide of HIV-1 reverse transcriptase andthe p51 subunit polypeptide of HIV-1 reverse transcriptase whichcomprises:

a) determining whether a compound is an activator of the formation ofthe complex between the p66 subunit polypeptide of HIV-1 reversetranscriptase and the p51 subunit polypeptide of HIV-1 reversetranscriptase according to a method which comprises: i) contacting ayeast cell cotransformed with a first plasmid which expresses in thecell a fusion protein comprising the p66 subunit polypeptide of HIV-1reverse transcriptase and a second plasmid which expresses in the cell afusion protein comprising the p51 subunit polypeptide of HIV-1 reversetranscriptase with the compound wherein the cell further comprises areporter gene which is activated in the presence of a complex betweenthe p66 subunit polypeptide and the p51 subunit polypeptide; ii)determining the level of activity of the reporter gene in the cell inthe presence of the compound; and iii) comparing the level of activityof the reporter gene determined in step (ii) with the level of activityof the reporter gene in the absence of the compound, wherein anincreased level of activity of the reporter gene indicates that thecompound is an activator of the formation of the complex between the p51subunit polypeptide of HIV-1 reverse transcriptase and the p66 subunitpolypeptide of HIV-1 reverse transcriptase; and

b) admixing the compound determined to be the activator in step (a)(iii)with a pharmaceutically acceptable carrier. Any of the above-describedmethods to determine whether a compound is an activator of the formationof the complex between the p66 subunit polypeptide of HIV-1 reversetranscriptase and the p51 subunit polypeptide of HIV-1 reversetranscriptase may be used in the method of making a pharmaceuticalcomposition comprising the determined activator compound, but is notlimited thereto, since one of skill will readily be able to substitutewell known reporter genes for the reporter genes used in the examplesherein. Moreover, one of skill is not limited to using the yeast cellsexemplified in any of the above-described methods herein, but may modifythe methods to use other eukaryotic cells, mammalian cells or cell linessuch as 298 T cells.

This invention further provides a method of testing a compound todetermine whether it is an inhibitor of formation of a complex between afirst p66 subunit polypeptide of HIV-1 reverse transcriptase and asecond p66 subunit polypeptide of HIV-1 reverse transcriptase whichcomprises: a) contacting a yeast cell cotransformed with a first plasmidwhich expresses in the cell a fusion protein comprising the first p66subunit polypeptide of HIV-1 reverse transcriptase and a second plasmidwhich expresses in the cell a fusion protein comprising the second p66subunit polypeptide of HIV-1 reverse transcriptase with the compoundwherein the cell further comprises a reporter gene which is activated inthe presence of a complex between the first p66 subunit polypeptide andthe second p66 subunit polypeptide; b) determining the level of activityof the reporter gene in the cell in the presence of the compound; and c)comparing the level of activity of the reporter gene determined in step(b) with the level of activity of the reporter gene in the absence ofthe compound, wherein a decreased level of activity of the reporter geneindicates that the compound is an inhibitor of the formation of thecomplex between the first p66 subunit polypeptide of HIV-1 reversetranscriptase and the second p66 subunit polypeptide of HIV-1 reversetranscriptase.

In an embodiment of the above-described method of testing a compound todetermine whether it is an inhibitor of formation of a complex between afirst p66 subunit polypeptide of HIV-1 reverse transcriptase and asecond p66 subunit polypeptide of HIV-1 reverse transcriptase, thefusion protein expressed by the first plasmid further comprises fulllength bacterial protein LexA fused to the first p66 subunit polypeptideof HIV-1 reverse transcriptase at amino acid position 1 of theN-terminal amino acid sequence of the full length bacterial protein LexAand the fusion protein expressed by second plasmid further comprisesamino acids at positions 768-881 of the C-terminal amino acid sequenceof Gal4AD fused to one end of an influenza hemagglutinin (HA) epitopeand a six alanine linker fused at a first end to the second end of theinfluenza hemagglutinin (HA) epitope, wherein the second p66 subunitpolypeptide of HIV-1 reverse transcriptase is fused at the second end ofthe six alanine linker.

This invention also provides a method of making a pharmaceuticalcomposition comprising an inhibitor of formation of a complex between afirst p66 subunit polypeptide of HIV-1 reverse transcriptase and asecond p66 subunit polypeptide of HIV-1 reverse transcriptase whichcomprises: a) determining whether a compound is an inhibitor offormation of a complex between a first p66 subunit polypeptide of HIV-1reverse transcriptase and a second p66 subunit polypeptide of HIV-1reverse transcriptase according to a method which comprises: i)contacting a yeast cell cotransformed with a first plasmid whichexpresses in the cell a fusion protein comprising the first p66 subunitpolypeptide of HIV-1 reverse transcriptase and a second plasmid whichexpresses in the cell a fusion protein comprising the second p66 subunitpolypeptide of HIV-1 reverse transcriptase with the compound wherein thecell further comprises a reporter gene which is activated in thepresence of a complex between the first p66 subunit polypeptide and thesecond p66 subunit polypeptide; ii) determining the level of activity ofthe reporter gene in the cell in the presence of the compound; and iii)comparing the level of activity of the reporter gene determined in step(ii) with the level of activity of the reporter gene in the absence ofthe compound, wherein a decreased level of activity of the reporter geneindicates that the compound is an inhibitor of the formation of thecomplex between the first p66 subunit polypeptide of HIV-1 reversetranscriptase and the second p66 subunit polypeptide of HIV-1 reversetranscriptase; and b) admixing the compound determined to be theinhibitor in step (a) (iii) with a pharmaceutically acceptable carrier.Any of the above-described methods to determine whether a compound is aninhibitor of formation of a complex between a first p66 subunitpolypeptide of HIV-1 reverse transcriptase and a second p66 subunitpolypeptide of HIV-1 reverse transcriptase may be used in the method ofmaking a pharmaceutical composition comprising the determined inhibitorcompound, but is not limited thereto, since one of skill will readily beable to substitute well known reporter genes for the reporter genes usedin the examples herein. Moreover, one of skill is not limited to usingthe yeast cells exemplified in any of the above-described methodsherein, but may modify the methods to use other eukaryotic cells,mammalian cells or cell lines such as 298 T cells.

The inhibitors determined by the above-described methods are useful forthe preparation of drugs, as pharmaceutical compositions, which willblock complex formation between the first p66 subunit polypeptide ofHIV-1 reverse transcriptase (homodimer)and the second p66 subunitpolypeptide of HIV-1 reverse transcriptase (homodimer so as to kill theHIV-1 virus (as well as formation of a complex between the p66 subunitpolypeptide of HIV-1 RT and the p51 subunit polypeptide of HIV-1 RT) orrender it inactive or inacabable of infecting cells of a subject,including a human subject.

This invention still further provides a method of testing a compound todetermine whether it is an activator of formation of a complex between afirst p66 subunit polypeptide of HIV-1 reverse transcriptase and asecond p66 subunit polypeptide of HIV-1 reverse transcriptase whichcomprises: a) contacting a yeast cell cotransformed with a first plasmidwhich expresses in the cell a fusion protein comprising the first p66subunit polypeptide of HIV-1 reverse transcriptase and a second plasmidwhich expresses in the cell a fusion protein comprising the second p66subunit polypeptide of HIV-1 reverse transcriptase with the compoundwherein the cell further comprises a reporter gene which is activated inthe presence of a complex between the first p66 subunit polypeptide andthe second p66 subunit polypeptide; b) determining the level of activityof the reporter gene in the cell in the presence of the compound; and c)comparing the level of activity of the reporter gene determined in step(b) with the level of activity of the reporter gene in the absence ofthe compound, wherein a increased level of activity of the reporter geneindicates that the compound is an activator of the formation of thecomplex between the first p66 subunit polypeptide of HIV-1 reversetranscriptase and the second p66 subunit polypeptide of HIV-1 reversetranscriptase.

In an embodiment of the above-described method of testing a compound todetermine whether it is an activator of formation of a complex between afirst p66 subunit polypeptide of HIV-1 reverse transcriptase and asecond p66 subunit polypeptide of HIV-1 reverse transcriptase, thefusion protein expressed by the first plasmid further comprises fulllength bacterial protein LexA fused to the first p66 subunit polypeptideof HIV-1 reverse transcriptase at amino acid position 1 of theN-terminal amino acid sequence of the full length bacterial protein LexAand the fusion protein expressed by second plasmid further comprisesamino acids at positions 768-881 of the C-terminal amino acid sequenceof Gal4AD fused to one end of an influenza hemagglutinin (HA) epitopeand a six alanine linker fused at a first end to the second end of theinfluenza hemagglutinin (HA) epitope, wherein the second p66 subunitpolypeptide of HIV-1 reverse transcriptase is fused at the second end ofthe six alanine linker.

The activators determined by the above-described methods are useful forthe preparation of drugs, as pharmaceutical compositions, which willenhance complex formation prematurely or inappropriately between thefirst p66 subunit polypeptide of HIV-1 reverse transcriptase and thesecond p66 subunit polypeptide of HIV-1 reverse transcriptase so as tokill the HIV-1 virus or render the the HIV-1 virus inactive or incapableof infecting cells of a subject, i.e. lack the functions of an infectedHIV-1 virus, including human subjects.

This invention also provides a method of making a pharmaceuticalcomposition comprising an activator of formation of a complex between afirst p66 subunit polypeptide of HIV-1 reverse transcriptase and asecond p66 subunit polypeptide of HIV-1 reverse transcriptase whichcomprises: a) determining whether a compound is an activator offormation of a complex between a first p66 subunit polypeptide of HIV-1reverse transcriptase and a second p66 subunit polypeptide of HIV-1reverse transcriptase according to a method which comprises: i)contacting a yeast cell cotransformed with a first plasmid whichexpresses in the cell a fusion protein comprising the first p66 subunitpolypeptide of HIV-1 reverse transcriptase and a second plasmid whichexpresses in the cell a fusion protein comprising the second p66 subunitpolypeptide of HIV-1 reverse transcriptase with the compound wherein thecell further comprises a reporter gene which is activated in thepresence of a complex between the first p66 subunit polypeptide and thesecond p66 subunit polypeptide; ii) determining the level of activity ofthe reporter gene in the cell in the presence of the compound; and iii)comparing the level of activity of the reporter gene determined in step(ii) with the level of activity of the reporter gene in the absence ofthe compound, wherein a increased level of activity of the reporter geneindicates that the compound is an activator of the formation of thecomplex between the first p66 subunit polypeptide of HIV-1 reversetranscriptase and the second p66 subunit polypeptide of HIV-1 reversetranscriptase; and b) admixing the compound determined to be theactivator in step (a) (iii) with a pharmaceutically acceptable carrier.Any of the above-described methods to determine whether a compound is anactivator of formation of a complex between a first p66 subunitpolypeptide of HIV-1 reverse transcriptase and a second p66 subunitpolypeptide of HIV-1 reverse transcriptase may be used in the method ofmaking a pharmaceutical composition comprising the determined activatorcompound, but is not limited thereto, since one of skill will readily beable to substitute well known reporter genes for the reporter genes usedin the examples herein. Moreover, one of skill is not limited to usingthe yeast cells exemplified in any of the above-described methodsherein, but may modify the methods to use other eukaryotic cells,mammalian cells or cell lines such as 298 T cells.

Methods of treating a subject infected with HIV-1 include administeringany of the above-described pharmaceutical compositions comprising: aninhibitor of the formation of the complex between the p66 subunitpolypeptide of HIV-1 reverse transcriptase; an activator of theformation of the complex between the p66 subunit polypeptide of HIV-1reverse transcriptase and the p51 subunit polypeptide of HIV-1 reversetranscriptase; an inhibitor of formation of a complex between a firstp66 subunit polypeptide of HIV-1 reverse transcriptase and a second p66subunit polypeptide of HIV-1 reverse transcriptase; and/or an activatorof formation of a complex between a first p66 subunit polypeptide ofHIV-1 reverse transcriptase and a second p66 subunit polypeptide ofHIV-1 reverse transcriptase. One of skill will recognize that otherpharmaceutical compositions may be administered to a subject infectedwith HIV-1 in conjunction with the pharmaceutical compositions providedby the methods set forth herein.

The invention also provides a pharmaceutical composition comprising aneffective amount of any of the above-described inhibitors and apharmaceutically acceptable carrier. The invention also provides apharmaceutical composition comprising an effective amount of any of theabove-described activators and a pharmaceutically acceptable carrier. Inthe subject invention an “effective amount” is any amount of aninhibitor or activator which, when administered to a subject sufferingfrom a disease or abnormality against which the inhibitors or activatorsare effective, causes reduction, remission, or regression of the diseaseor abnormality. In the practice of this invention the “pharmaceuticallyacceptable carrier” is any physiological carrier known to those ofordinary skill in the art useful in formulating pharmaceuticalcompositions.

In one preferred embodiment the pharmaceutical carrier may be a liquidand the pharmaceutical composition would be in the form of a solution.In another equally preferred embodiment, the pharmaceutically acceptablecarrier is a solid and the composition is in the form of a powder ortablet. In a further embodiment, the pharmaceutical carrier is a gel andthe composition is in the form of a suppository or cream. In a furtherembodiment the compound or composition may be formulated as a part of apharmaceutically acceptable transdermal patch.

A solid carrier can include one or more substances which may also act asflavoring agents, lubricants, solubilizers, suspending agents, fillers,glidants, compression aids, binders or tablet-disintegrating agents; itcan also be an encapsulating material. In powders, the carrier is afinely divided solid which is in admixture with the finely dividedactive ingredient. In tablets, the active ingredient is mixed with acarrier having the necessary compression properties in suitableproportions and compacted in the shape and size desired. The powders andtablets preferably contain up to 99% of the active ingredient. Suitablesolid carriers include, for example, calcium phosphate, magnesiumstearate, talc, sugars, lactose, dextrin, starch, gelatin, cellulose,polyvinylpyrrolidine, low melting waxes and ion exchange resins.

Liquid carriers are used in preparing solutions, suspensions, emulsions,syrups, elixirs and pressurized compositions. The active ingredient canbe dissolved or suspended in a pharmaceutically acceptable liquidcarrier such as water, an organic solvent, a mixture of both orpharmaceutically acceptable oils or fats. The liquid carrier can containother suitable pharmaceutical additives such as solubilizers,emulsifiers, buffers, preservatives, sweeteners, flavoring agents,suspending agents, thickening agents, colors, viscosity regulators,stabilizers or osmo-regulators. Suitable examples of liquid carriers fororal and parenteral administration include water (partially containingadditives as above, e.g. cellulose derivatives, preferably sodiumcarboxymethyl cellulose solution), alcohols (including monohydricalcohols and polyhydric alcohols, e.g. glycols) and their derivatives,and oils (e.g. fractionated coconut oil and arachis oil). For parenteraladministration, the carrier can also be an oily ester such as ethyloleate and isopropyl myristate. Sterile liquid carriers are useful insterile liquid form compositions for parenteral administration. Theliquid carrier for pressurized compositions can be halogenatedhydrocarbon or other pharmaceutically acceptable propellent.

Liquid pharmaceutical compositions which are sterile solutions orsuspensions can be utilized by for example, intramuscular, intrathecal,epidural, intraperitoneal or subcutaneous injection. Sterile solutionscan also be administered intravenously. The compounds may be prepared asa sterile solid composition which may be dissolved or suspended at thetime of administration using sterile water, saline, or other appropriatesterile injectable medium. Carriers are intended to include necessaryand inert binders, suspending agents, lubricants, flavorants,sweeteners, preservatives, dyes, and coatings.

The inhibitor(s) or activator(s) determined by the methods describedabove can be administered orally in the form of a sterile solution orsuspension containing other solutes or suspending agents, for example,enough saline or glucose to make the solution isotonic, bile salts,acacia, gelatin, sorbitan monoleate, polysorbate 80 (oleate esters ofsorbitol and its anhydrides copolymerized with ethylene oxide) and thelike.

The inhibitor(s) or activator(s) can also be administered orally eitherin liquid or solid composition form. Compositions suitable for oraladministration include solid forms, such as pills, capsules, granules,tablets, and powders, and liquid forms, such as solutions, syrups,elixirs, and suspensions. Forms useful for parenteral administrationinclude sterile solutions, emulsions, and suspensions.

Optimal dosages to be administered may be determined by those skilled inthe art, and will vary with the particular inhibitor(s) or activator(s)in use, the strength of the preparation, the mode of administration, andthe advancement of the disease condition or abnormality. Additionalfactors depending on the particular subject being treated will result ina need to adjust dosages, including subject age, weight, gender, diet,and time of administration.

This invention will be better understood from the Experimental Detailsthat follow. However, one skilled in the art will readily appreciatethat the specific methods and results discussed are merely illustrativeof the invention as described more fully in the claims that followthereafter.

Experimental Details

First Series of Experiments

Materials and Methods

Bacterial and Yeast Strains

Saccharomyces cerevisiae strain CTY10-5d (MATa ade2 trpl-901 leu2-3, 112his3-200 gal4-gal80-URA3::lexA-lacZ) contains an integrated GAL1-lacZgene with the lexA operator (a gift from Stanley Fields, StateUniversity of New York, Stony Brook). The yeast strain HF7c containsCYC1-lacZ gene with three copies of the GAL4 responsive UASG 17-meroperator (CLONTECH). Escherichia coli mutator strain XL1-Red(Stratagene) was used for random mutagenesis whereas XL1-Blue(Stratagene) was used to amplify the mutated library. KC8 (CLONTECH), anauxotrophic leuB, trpC and hisB E. coli strain, was used to isolateplasmids from yeast. E. coli strains M15 and BL21 were used to expressp66-His and glutathione S-transferase-tagged p51 (GST-p51) respectively(see below).

Yeast Methods

Transformation of yeast and the qualitative β-galactosidase (β-gal)colony lift assay were as published (19). Quantification ofprotein-protein interactions was determined using the β-gal liquid assayperformed on permeabilized yeast grown from three independenttransformants using orthonitrophenyl-β-D-galactopyranoside as substrate(19).

Protein Expression and RT Activity

Fusion protein expression in yeast was determined by Western blot oflysates with Gal4AD polyclonal antibodies (Upstate Biotechnology, LakePlacid, N.Y.), anti-lexA polyclonal antibodies (Invitrogen) and HIV-1 RTpolyclonal (Intracel, Cambridge, Mass.) or 5B2 monoclonal antibody (20).Immunodetection was with ECL-Plus (Amersham) To measure RT activity,yeast lysates were prepared by glass bead disruption (19) and enzymeactivity was determined in exogenous assays (21) and quantified byphosphoimager analysis.

Yeast Shuttle Vectors

pSH2-1 (22) and pLex202-PL (23) express the lexA DNA binding domain(lexA₈₇) and the full-length lexA protein (lexA₂₀₂), respectively. pGBT9and pAS2-1, both containing the GAL4 DNA binding domain (GAL4 BD), werepurchased from CLONTECH. pNLexA allows expression of proteins fused tothe N terminus of full-length lexA₂₀₂ (OriGene Technologies, Rockville,Md.). pGADNOT (18) and pACTII (24) allow expression of proteins fused tothe Gal4 activation domain (GAL4 AD). pACTII also contains the influenzahemagglutinin (HA) epitope tag located between GAL4AD and thepolylinker.

Construction of HIV-1 RT Fusions in Yeast Vectors

Constructs and expressed fusion proteins are as described in FIG. 1. TheRT sequence for constructing the following expression vectors wasamplified from HIV-1 molecular clone pNLenv-1 (containing the HIVNL43sequence) (25). The p66 amplimers were cloned into the BamHI-SalI sitesof pGBT9, pSH2-1, pLex202-PL, pACTII and pGADNOT; the BamH1-XhoI sitesof pACTII; and the EcoRI-BamHI sites of pNLexA. p51 amplimers werecloned into the BamHI-SalI sites of these vectors except for cloninginto pACTII, where the BamH1-XhoI sites were used. The HXB2 RT sequencefrom pHXB2gpt (26) was used to construct p66HXAlaLex202 and p51HXGADNOT.

Construction of HIV-1 RT Deletion Mutants

All p66 deletion mutants were prepared by cloning PCR amplimers into theBamHI-SalI sites of pSH2-1. Fingers, palm, connection, thumb and RNase Hdomains of HIV-1 RT are denoted F, P, C, T and R respectively.pT+C+RSH2-1 (encoding lexA₈₇-T+C+R) contains RT (from HIVNL43) codons236-560. pC+RSH2-1 (encoding lexA871-C+R) contains codons 322-560 whilepRSH2-1 (encoding lexA₈₇-R) comprises codons 425-560. All p51 deletionmutants were prepared by cloning of PCR amplimers into the BamHI-XhoIsites of pACTII. pF+P+T−ACTII (encoding Gal4AD−HA−F+P+T) includes RTcodons 1-325 and pF+P−ACTII (encoding Gal4AD−HA−F+P) has codons 1-244.p51Δ13ACTII (encoding Gal4AD-HA-51Δ13) contains RT codons 1-426.p51Δ26GADNOT (encoding Gal4AD-51Δ26) was obtained by random mutagenesisof p55GADNOT in XL1-Red.

Construction of RT Fusions with the L234A Mutation and RandomMutagenesis of p66Ala234Lex202 and Selection of Revertants

p66Ala234Lex202 (encoding lexA₂₀₂-Ala-66L234A) was made by inserting p66from p6HprotL234A (a gift from Vinayaka Prasad, Albert Einstein Collegeof Medicine, Bronx N.Y.) into the BamHI/SalI sites of pLex202-PL.p51234GADNOT (encoding Gal4AD-51L234A) was made by insertion of p51 fromp6HprotL234A into the BamHI-SalI sites of pGADNOT. Second-site mutationsrestoring dimerization to lexA₂₀₂-Ala-66L234A were generated bypropagation of p66Ala234Lex202 in XL1-Red (Stratagene). Two independentpools were prepared. CTY10-5d was cotransformed with the mutagenizedlibrary and either p51234GADNOT or p51GADNOT. Blue colonies were pickedfrom β-gal colony lift assays and clonally purified. p66 DNA fromisolated plasmids were recloned into a nonmutated pLex202-PL backboneand reintroduced into CTY10-5d to confirm the phenotype. Mutationspresent in p66 were determined by automated nucleotide sequencing.

Site Directed Mutagenesis

p66 with genotype D110G was prepared from a p66 clone containing bothD110G and L234A obtained by random mutagenesis by backmutation of codon234 to wild-type. p66 with either the W402R or W406R substitutions wereprepared by subcloning a Bsp1286I-SalI fragment (600 bp) from the clonesobtained by random mutagenesis of L234A with wild-type BamHI-Bsp1286Ifragment (1,080 bp) from p66HXAlaLex202 into pLex202-PL.

In Vitro Heterodimerization

Plasmids expressing wild-type and p66 mutants with a histidine tag atthe C-terminus (p66-His) were constructed by cloning the p66 codingregion into the SphI-BglII site of pQE-70 (Qiagen, Chatsworth, Calif.).The C-terminal tag was appended as described previously [clone 3 (27)].Glutathione S-transferase tagged p51 (GST-p51) was prepared bysubcloning the BamHI-SalI fragment from p51HXGADNOT into pGEX5X-3(Amersham Pharmacia). Cells were induced and then lysed by the additionof 1 mg/ml of lysozyme to 1 ml of lysis buffer [50 mM sodium phosphatebuffer (pH 7.8), 500 mM NaCl, 0.5% Nonidet P-40, 5 mM DTT, and 1 μg/mleach of pepstatin A, aprotinin and leupeptin] and clarified. Lysateswere combined and incubated for 16 hrs at 4° C. The heterodimer wascaptured on Glutathione Sepharose 4B beads and unbound subunits wereremoved by washing with lysis buffer. Heterodimer bound to beads wereresolved by SDS/PAGE. For quantification of RT activity, dimers wereeluted from beads with 10 mM reduced glutathione in 50 mM Tris (pH 8.0).Samples were assayed for DNA polymerase activity on homopolymerictemplate-primers for various times, and the activity was determined fromthe initial slope of the linear phase of the time course. Western blotconfirmed equal recovery of GST-51 protein in each sample.

RESULTS

Expression of RT Fusion Proteins and RT Activity

The stable expression of p66 was tested in several contexts, as eitherGal4BD or LexA fusions, and using a six alanine linker to separate p66from its fusion partner. p66 fused to the C terminus of lexA₈₇, the C orN termini of lexA₂₀₂ (with or without a six alanine spacer), and in avariety of contexts to the Gal4AD were all stably expressed (FIG. 1). Incontrast, p66 fused to the C terminus of the Gal4BD (Gal4BD-66) was notexpressed in yeast at detectable levels (FIG. 1). The smaller RTsubunit, p51, was well expressed as fusions with the Gal4BD, Gal4AD, andboth lexA₈₇ and lexA₂₀₂. We examined whether the bait fusions encoded byp66SH2-1, p66AlaLex202 and p66NLexA exhibited RT activity in yeast. Allthree fusion proteins demonstrated high levels of RT activity comparedwith protein lysates from yeast transformed with an empty vector (datanot shown). These data suggest that the p66 fusion proteins arefunctional and in a conformation consistent with measurable catalyticactivity.

Heteromeric Interactions of p66 and p51 by Transactivation in theTwo-Hybrid System

To test whether the Y2H system could detect the interaction of thep66/p51 heterodimer, we cotransformed yeast reporter strains withplasmids expressing p66DNA BD and p51DNA AD fusion proteins (Table 1).β-gal activity expressed in yeast, which indicates the strength of theinteraction between the fusion proteins, was assessed by bothqualitative and quantitative assays. The p66 bait fusions expressed fromp66SH2-1, p66AlaLex202 and p66NLexA interacted with Gal4AD-p51 domainfusions (Table 1) but not with Gal4AD alone (Table 1). The strongestinteractions were observed with p66 baits lexA₂₀₂-Ala-66 and 66-lexA₂₀₂.Moreover, p51 expressed in pACTII gave a stronger signal than p51GADNOTwhen coexpressed with p66 fusion baits. Despite the stable expression ofthe p66 fusion protein, lexA₂₀₂-66, no significant interaction with p51was detected (FIG. 1). Moreover, lexA₂₀₂-66 yielded the same weak signalwith the empty Gal4AD vector, pGADNOT, indicating that this version ofp66 is weakly self-activating even without a partner.

TABLE 1 Interaction of p66 binding domain fusions with p51 activationdomain fusions in the Y2H system β-gal activity Constructs OperatorColony* Liquid† p66SH2-1 + pGADNOT lexA − ND p66SH2-1 + pACTII lexA −0.02 p66SH2-1 + p51GADNOT lexA ++ 0.5 p66SH2-1 + p51ACTII lexA +++ 3.5p66AlaLex202 + pGADNOT lexA − ND p66AlaLex202 + pACTII lexA − 0.04p66AlaLex202 + p51GADNOT lexA +++ 1.6 p66AlaLex202 + p51ACTII lexA +++7.7 p66NLexA + pGADNOT lexA − 0.06 p66NLexA + pACTII lexA − 0.04p66NLexA + p51GADNOT lexA +++ 6.6 p66NLexA + p51ACTII lexA +++ 25.0p66Lex202 + pGADNOT lexA +/− ND p66Lex202 + p51GADNOT lexA +/− NDp66GBT9 + pGADNOT‡ UAS_(G) − ND p66GBT9 + p51GADNOT‡ UAS_(G) − ND Yeaststrain CTY10-5d or ‡HF7c were transformed with plasmids encoding p66bait and p51 prey fusions. Fusion proteins encoded by plasmids aredescribed in Materials and Methods and FIG. 1. *Transformants werelifted onto nitrocellulose and subjected to the β-gal colony lift assayto determine intensities of blue color produced; +++, strong blue in 1h; ++, blue in 1 h; +/−, weak blue in 3 h; −, white; ND, not done.

We also showed that heteromeric interactions between p66 and p51 couldbe detected in the reciprocal configuration with p51 as either a LexA orGal4BD fusion and p66 as a Gal4AD fusion (Table 2). The demonstration ofheteromeric dimerization of p66 and p51 in different contexts stronglysuggests that the interaction is specific. Tests for interaction withfive unrelated proteins showed no signal (data not shown), providingfurther evidence for the specificity of RT heterodimerization in yeast.

TABLE 2 Interaction of p51 binding domain fusions with p66 activationdomain fusions in the Y2H system β-gal activity Constructs OperatorColony* Liquid p51SH2-1 + pGADNOT lexA − 0.06 p51SH2-1 + pACTII lexA −0.05 p51SH2-1 + p66AlaACTII lexA ++ 1.2 p51Lex202 + pACTII lexA − 0.05p51Lex202 + p66AlaACTII lexA +++ 3.2 p51AS2-1 + pACTII‡ UAS_(G) − NDP51AS2-1 + P66AlaACTII‡ UAS_(G) ++ ND Yeast strain CTY10-5d or ‡HF7cwere transformed with plasmids encoding p51 bait and p66 prey fusions.Fusion proteins encoded by plasmids are described in Materials andMethods and FIG. 1. *As defined in Table 1. ‡As defined in Table 1.

Homomeric Interactions

The interaction of the RT heterodimer p66/p51 has a dissociationconstant of 10⁻⁹ M, whereas the dissociation constants for the p66 andp51 homodimers are 10⁻⁶ M and 10⁻⁵ M, respectively (9). We were unableto detect p51 homodimerization when CTY10-5d was cotransformed witheither p51SH2-1 or p51Lex202 baits and p51ACTII prey (data not shown).In contrast, p66 homodimerization could be detected when yeast wascotransformed with p66NlexA bait and p66AlaACTII prey (β-gal activity0.3 Miller units). p66 homodimerization of these two constructs was100-fold weaker compared to the interaction of p66NlexA with p51ACTII(Table 1). The strength of the interactions observed in vivo areconsistent with biochemical data.

p66 Domains that Interact with p51

We used the Y2H RT dimerization assay to map the regions of p66 requiredfor binding to p51 (FIG. 2). A series of mutants with sequentialdeletions in the polymerase subdomains were prepared as C-terminalfusions with lexA₈₇. Deletion of the fingers and palm subdomains(lexA₈₇−T+C+R) did not significantly affect binding to Gal4AD-HA-51. Afurther deletion of the thumb subdomain (lexA87−C+R) resulted in reducedβ-gal activity (FIG. 2). Expression of the RNase H domain alone was notsufficient for interaction with p51. This lack of interaction was notattributable to an aberrant RNase H conformation, as lexA₈₇-R alsointeracted as strongly as lexA₈₇-66 with a cellular protein, diaphorase,that we find interacts with the RNase H domain of RT in the Y2H system(results not shown). None of the bait fusions demonstrated activation ofthe lacZ reporter gene when coexpressed with Gal4AD-HA alone, excludingthe possibility of nonspecific self-activation by the bait fusions(results not shown). These data suggest that the connection and RNase Hsubdomains of p66 are sufficient for interaction with p51.

The C Terminus of p51 is Required for Interaction with p66

It has previously been shown biochemically that deletion of as little as25 amino acids from the C terminus of p51 can prevent dimerization top66 (15). To ascertain whether this effect could be observed underphysiological conditions in the Y2H system, we constructed a series ofC-terminal deletion mutants of p51 prey fusions and assayed interactionwith p66 bait. Deletion of 13 amino acids from the C terminus of p51 hadlittle effect (1.8-fold decrease) on dimerization with p66 (FIG. 3).However, deletions of 26 amino acids and greater abrogated RTdimerization, indicating the importance of the C-terminal 26 amino acidsof p51 in these interactions. These results also suggest that the systemfaithfully recapitulates the behavior of the enzyme as studied in vitro.

L234A in p66 Subunit Inhibits RT Dimerization

The Y2H RT dimerization assay would be most useful if it could beapplied to the analysis of single amino acid substitutions that affectheteromeric interactions. To test the system, we introduced the L234Aprimer grip mutation, previously shown biochemically to inhibit p66/p51association (10), into both RT subunits. The presence of L234A in bothp66 and p51 totally inhibited RT dimerization as observed by a 53-folddecrease in the β-gal signal compared to wild-type proteins (FIG. 4). Toassess the effect of L234A in individual subunits CTY10d-5 wascotransformed with constructs expressing either p66 mutant bait andwild-type p51 prey, or p66 wild-type bait with psi mutant prey. Lessthan a two-fold decrease in the signal compared with wild-type fusionswas observed when the L234A mutant p51 (Gal4AD-51L234A) was coexpressedwith the wild-type fusion leXA₂₀₂-Ala-66 (FIG. 4). However, a 32-foldinhibition was observed for the interaction of the mutantlex₂₀₂-Ala-66L234A with wild-type Gal4AD-51. These data suggest thatL234A affects dimerization predominantly through p66, as has beenpreviously reported (10). Analysis of fusion protein expression in yeastby Western blot analysis revealed that all fusion proteins, includingthe L234A mutants, were stably expressed (results not shown).

Second-Site Mutations that Restore Heterodimerization and RT Activity tothe p66L234A Mutant

To gain insight into the mechanism of inhibition of RT dimerization byL234A, we attempted to select for second-site suppressor mutations inp66 that restore dimerization with p51. To select for p66 mutants withrestored dimerization, CTY10-5d was cotransformed with a librarygenerated by mutagenesis of p66AlaL234ALex202 and a plasmid expressingeither Gal4AD-51 or the Gal4AD-51-L234A mutant. A total of 25,000colonies from each of two independently mutated libraries were screened.Six and five blue colonies were obtained when lex₂₀₂-Ala-66L234A wascotransformed with Gal4AD-51 and Gal4AD-51-L234A, respectively. CTY10-5dwas retransformed with each isolated library plasmid and with eitherp51HXGADNOT or p51L234AGADNOT; the recovered clones showed restoredbinding activity with both p51 fusion proteins. Five types of mutationswere observed (Table 3). Single amino acid changes in the clones thatretained the L234A change included D110G, D186V, W402R and W406R. Theremaining three clones had reverted to wild-type at codon 234 (Table 3).

TABLE 3 Second site mutations in lexA₂₀₂66HXL234A that restoredimerization to p51 β-gal activity Constructs Number of Clones Colony*Liquid† Wild Type NA +++ 3.2 L234A NA − 0.1 D110G NA +++ 6.9 W402R NA+++ 7.1 W406R NA +++ 5.8 L234A; D110G 3 +++ 1.5 L234A; D186V 1 +/− 0.2L234A; W402R 3 +++ 7.1 L234A; W406R 1 +++ 6.1 L234 3 +++ 4.7 Yeaststrain CTY10-5d was cotransformed with p51HXGADNOT and various clonesexpressing lexA₂₀₂-Ala-66HX fusions with mutations in p66 as indicated.NA, not applicable. *As defined in Table 1. ‡As defined in Table 1.

Two of the changes are at the catalytically essential aspartyl residuesD110 and D186. These residues are not located at the dimer interface,and mutations at these residues result in an inactive RT (28) (FIG. 5).A variant p66 containing D110G alone, without L234A, gave a 2-foldstronger β-gal signal than wild-type p66 for heterodimerization and was4.6-fold stronger compared with clones containing both L234A and D110G.Partial restoration of dimerization by D110G suggests thatconformational changes at the active site compensate for structuralchanges mediated by L234A.

The second set of mutations, W402R and W406R, are located at the dimerinterface (FIG. 5) in a tryptophan repeat region which is highlyconserved among HIV-1, HIV-2 and closely related simian immunodeficiencyvirus RTs (29). In the L234A genetic background, these mutationsresulted in a dramatic increase in the β-gal signal over the parent andyielded a 2-fold higher signal for heterodimerization compared withwild-type RT fusions (Table 3). W402R and W406R in a wild-type geneticbackground had the same enhanced β-gal activity as the restored mutants(Table 3). Therefore, the mutations in the tryptophan repeat motif mayenhance the interaction with GAL4AD-51 independently of the L234Amediated defect.

To confirm that the second-site mutations could restoreheterodimerization to the L234A parent in an alternative assay, weexamined the binding of these p66 mutants to p51 in vitro. Bacteriallysates containing GST-p51 or wild-type and mutant p66-His wereincubated together, and heterodimers were captured on GlutathioneSepharose 4B beads. As expected, wild-type p66 dimerized with GST-p51whereas the p66L234A mutant did not (FIG. 6A). Restoration ofdimerization by D110G, W402R or W406R in the L234A parent was observed(FIG. 6A), thus confirming our observations in the Y2H assay.

To determine whether restoration of heterodimerization was associatedwith enhanced DNA polymerase activity, heterodimers eluted from beadswere assayed for RT activity (FIG. 6C). GST-p51 had significantbackground activity compared with wild-type enzyme. The enzyme resultingfrom incubation with p66L234A had the same background activity. Asexpected, heterodimers comprising p66L234A containing the active sitemutation D110G also had only background activity. Interestingly, bothW402R and W406R mutations not only restored heterodimerization to theL234A parent but also increased RT activity, even above levels of thewild-type control (FIG. 6C).

Discussion

In this study we have shown that fusions of p66 and p51 can be stablyexpressed in yeast and can heterodimerize in reciprocal configurations.The presence of spacers in the form of alanine or an HA tag may havebeen an important aspect for stronger interactions in the Y2H assay.Moreover, we have validated the Y2H assay by comparing previouslydescribed effects of p51 deletions and the L234A substitution onheterodimerization. We have also shown how this assay can further thestudy of the HIV-1 RT structure-function by the identification ofsecond-site mutations that restore RT dimerization.

The palm, connection, and RNase H domains of p66 make major contactswith p51. An indication that the palm region is important is thedestabilization of the p66/p51 heterodimer by the nonnucleoside RTinhibitor 2′,5′-bis-O-(tert-butylidimethylsilyl)-3′-spiro-5″-4″-amino-1′,2″-oxothiole-2″,2″-dioxide)]-b-D-pentofuranosyl (TSAO) by itsinteraction between the palm subdomain of p66 and the β7-β8 loop in thefingers subdomain of p51 (30, 31). Preliminary tests of the addition ofTSAO to our in vitro binding assays confirm the ability of the drug toreduce heterodimerization (data not shown). Tests of the related drugTSAOe³T showed a more modest destabilization only detectable in thepresence of denaturants (31). Deletion mapping of the p66 domainsrequired for interaction with p51 suggests that the presence of theconnection and RNase H domains are sufficient for interaction with p51in the Y2H system. It is surprising that the deletion of the palm domainhad little effect on binding to p51 as this p66 subdomain provides amajor contact with p51 (9); however, the connection and RNase H domainsmay provide a sufficient surface for saturating the signal in yeast.

Truncation of the C terminus of p51 revealed that a 13-amino aciddeletion had little effect on dimerization with p66, but a deletion of26 amino acids abrogated heterodimerization as seen in the Y2H assay.These data are consistent with previous in vitro studies (15). AllC-terminal truncation mutants were stably expressed in yeast, excludingthe possibility of decreased expression affecting the signal. It ispossible that these C-terminal residues may have a direct role indimerization; or the deletion of these residues may effect thestructural integrity or correct positioning of the structural elementsα-L and β-20 (5,15). These elements contain the tryptophan repeat motif,which has been proposed to play an important role in HIV-1 dimerization(29, 32).

We have shown that the L234A substitution inhibits RT dimerization inyeast most dramatically when present on the p66 subunit of HIV-1 RT, aspreviously seen in vitro (10). L234A is located in the primer gripregion of p66 (5) and is highly conserved among avian, primate andmurine RTs (33). To help determine the mechanism by which L234A affectsheterodimerization, we selected for second-site mutations restoringp66/p51 association. Aside from clones which had reverted to thewild-type L234, we observed two classes of mutants: those withalterations either in the tryptophan repeat or in the polymerase activesite (FIG. 5). Both classes of suppressors were also shown to restorebinding of the mutant p66 subunit to p51 as measured in an in vitrobinding assay (FIG. 6A). L234A is not at the dimer interface, and it hasbeen proposed that it affects dimerization by indirectly affectingcontacts between P95 in the palm of p66 with residues in the β7-β8 loopof p51 (11). The mutations W402R and W406R are distant from this region,being located in the connection subdomain which contacts the p51connection domain in the heterodimer. The appearance of a basic residuein both codon 402 and 406 suggests a charge interaction with an acidicresidue in p51 or alternatively an increase in electrostatic potentialbetween the surfaces at the connection domain interface.

The recovery of second-site suppressor mutations at the catalyticallyessential aspartyl residues suggests that there is a relationshipbetween dimerization and active site residues. Neither D186V nor D110Gmake obvious contacts with L234A, although both are in the same palmsubdomain (FIG. 5)(2). Interaction between the NNRTI binding site, whichincludes L234, and the RT catalytic site has been suggested by bothstructural and enzymatic data explaining the mechanism of resistance toNNRTIs (34, 35). The D110G or D186V changes would probably result inloss of one of the two magnesium ions bound to the active site (36). Aloss of chelated magnesium in addition to a glycine change at 110 maylead to increased flexibility in that region, thus affectingdimerization. Determination of the crystal structure of the D110G RTmutant will help resolve these issues.

Heterodimerization of HIV-1 has been suggested as a target forchemotherapeutic intervention (7). To date, there are no HIV-1 RTdimerization inhibitors being used in the clinic. Nevertheless, thereare several reports of HIV-1 and HIV-2 RT dimerization inhibitors basedon peptides representing the conserved tryptophan repeat region of RT(32, 37). These peptides have been shown to prevent the association ofp66/p51 (32) and have demonstrable in vitro anti-HIV-1 activity (37).TSAO has been shown to destabilize the p66/p51 heterodimer and mayrepresent a nonpeptide RT dimerization inhibitor (30). In preliminarytests of this drug for its effects on heterodimerization in the Y2Hsystem, we saw no inhibition of β-gal activity (data not shown).However, the possibility that the drug is not taken up by yeast cannotbe ruled out. The availability of a Y2H assay for RT dimerization willfacilitate the screening for other such inhibitors of this processaccording to the methods set forth herein.

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SECOND SERIES OF EXPERIMENTS

Nonnucleoside reverse transcriptase inhibitors (NNRTIs) are allostericinhibitors of the human immunodeficiency virus type 1 (HIV-1) reversetranscriptase (RT). Yeast grown in the presence of many of these drugsexhibited dramatically increased association of the p66 and p51 subunitsof the HIV-1 RT as reported by a yeast two-hybrid assay. The enhancementrequired drug binding by RT; introduction of a drug-resistance mutationinto the p66 construct negated the enhancement effect. The drugs couldalso induce heterodimerization of dimerization defective mutants.Coimmunoprecipitation of RT subunits from yeast lysates confirmed theinduction of heterodimer formation by the drugs. In vitro bindingstudies indicate that NNRTIs can bind tightly to p66 but not p51, andthen mediate subsequent heterodimerization. This study demonstrates anovel effect of NNRTIs on the assembly of RT subunits.

The human immunodeficiency virus type 1 (HIV-1) reverse transcriptase(RT) catalyzes the conversion of genomic RNA into double-strandedproviral DNA after cell entry (1), utilizing the RNA- and DNA-dependentpolymerase and ribonuclease H (RNase H) activities of the enzyme. TheHIV-1 RT is an asymmetric dimer consisting of p66 and p51 polypeptides(2, 3). The p51 subunit contains the identical N-terminal sequences asp66, but lacks the C-terminal RNase H domain. The structure of the HIV-1RT has been elucidated by X-ray crystallography in several formsincluding the unliganded enzyme (4), in complex with nonnucleosidereverse transcriptase inhibitors (NNRTIs) (5, 6) and bound totemplate-primer with (7) or without dNTP substrate (8). The polymerasedomain of the p66 subunit resembles a right hand and contains thefingers, palm, thumb and connection subdomains, with the latter actingas a tether between the polymerase and RNase H regions (5, 8). Althoughp51 has the same polymerase domains as p66, the relative orientations ofthese individual domains differ markedly (5, 8). Structural analysisreveals three major contacts between p66 and p51, with most of theinteraction surfaces being hydrophobic (9, 10).

NNRTIs are chemically diverse, largely hydrophobic compounds whichcomprise over 30 different classes (11, 12). NNRTIs do not requireintracellular metabolism for activity, are noncompetitive inhibitors ofRT activity with respect to dNTP substrate and template/primer, and arerelatively noncytotoxic (11). NNRTIs bind to a hydrophobic pocket closeto but distinct from the polymerase active site in the p66 subunit (13,14) and inhibit enzyme activity by mediating allosteric changes in theRT (15, 16). Initial clinical use of NNRTIs as monotherapy and selectionof drug-resistant variants in cell culture results in the rapidemergence of highly drug-resistant variants due to single amino acidchanges (17, 18) in the NNRTI binding pocket that directly affect drugbinding (13, 14). The NNRTIs currently approved for use in highly activeantiretroviral therapy include nevirapine (19), delavirdine (20) andefavirenz (21). We have previously shown that HIV-1 RTheterodimerization can be effectively monitored in the yeast two-hybrid(Y2H) system using appropriately engineered constructs (22). We usedthis system to assess the effect of NNRTIs on the b-galactosidase(b-gal) readout in yeast. Several NNRTIs induced dramatic increases inb-gal activity and this increase was due to enhanced association betweenthe RT subunits as a result of a specific interaction of drug with thep66 subunit. These data document a novel effect of NNRTIs on HIV-1 RTdimerization and demonstrate that these drugs behave in a manner similarto chemical inducers of dimerization (CID), compounds that bind to atarget protein and promote an interaction with another protein (23).

Materials and Methods

Antiviral drugs

The drugs used in this study were: carboxanilides UC781, UC10, UC38,UC84 (24, 25), Uniroyal Chemical Ltd (Middlebury, Conn.); efavirenz(EFV) (21), DuPont Merck (Wilmington Del.); delavirdine (BHAP) (26),Pharmacia and Upjohn (Kalamazoo, Mich.); nevirapine (19), RoxanneLaboratories (Redding Conn.); HBY 097 (27), Hoechst-Bayer (Frankfurt,Germany); 8-chloro-TIBO (8-Cl-TIBO) (28) and a-APA (29), JanssenResearch Foundation (Beerse, Belgium). All drugs were dissolved indimethyl sulfoxide at a concentration of 10 mg/ml for use in Y2H and invitro assays.

Yeast and Bacterial Strains and Yeast Methods

Yeast and bacterial strains were as described previously (22).Transformation of yeast, the qualitative b-gal colony lift assay and thequantitative b-gal liquid assay were as previously described (22).

Construction of HIV-1 RT Fusions in Yeast Expression Vectors

The construction of p66SH2-1, p51SH2-1, p66GADNOT, p51GADNOT andp51ACTII which express the wild type p66 and p51 fusion proteinslexA₈₇-66, lexA87-51, Gal4AD-66, Gal4AD-51 and Gal4AD-HA-51,respectively were as described previously (22). p66L234ASH2-1 (encodinglexA₈₇-66L234A) was made by cloning the PCR amplification product fromthe RT region of p66AlaL234ALex202 (22) into the BamHI-SalI sites ofpSH2-1. p51234ACTII (encoding Gal4AD-HA-51L234A) was constructed bysubcloning the p51 BamHI-SalI fragment from p51234GADNOT (22) intopACTII. p66W401ASH2-1 (encoding lexA₈₇-66W401A), p51W401AACTII (encodingGal4AD-HA-51W401A) and p51W401AGADNOT (encoding Gal4AD-51W401A) weremade by PCR amplification of the RT region from plasmid pALRT-78S(A402)(a gift from John McCoy) and cloned into the BamHI-SalI sites of pSH2-1,pGADNOT or the BamHI-XhoI sites of PACTII. p66Y181CSH2-1 containing theY181C mutation in p66 of the lexA₈₇-66 fusion protein was prepared bysite-directed mutagenesis using the Gene Editor Kit (Promega, Madison,Wis.) according to the manufacturer's protocol.

Construction of HIV-1 RT Fusions in Bacterial Expression Vectors

Wild-type and p66 mutants (either L234A or W401A) were cloned into theSphI-BglII site of pQE-70 (Qiagen, Chatsworth, Calif.) (22). GlutathioneS-transferase-tagged p51 (GST-p51) and mutants containing either theW401A or L234A substitutions were constructed by cloning the p51encoding fragments into the BamHI-SalI site of pGEX5X-3 (AmershamPharmacia Biotech) (22).

Y2H RT Heterodimerization Assays for Measuring Effect of NNRTIs on B-GalActivity.

CTY10-5d transformed constructs expressing p66 bait and p51 prey fusionswere grown overnight to stationary phase in synthetic complete mediumwithout histidine and leucine and containing 2% glucose (SC-His-Leu).2.5 ml of media with or without drug were inoculated with 0.0125-0.25OD₆₀₀ units of CTY10-5d. Yeast were grown with aeration at 30° C. toOD₆₀₀=0.5. The equivalent of 1 OD₆₀₀ unit was pelleted for eachtreatment and subjected to a quantitative b-gal liquid assay.

Coimmunoprecipitation of p66 and p51 in Yeast Lysates.

Cultures (30 ml) containing no drug, efavirenz or UC781 and 0.1 OD₆₀₀units/ml of CTY10-5d expressing p66 bait and p51 prey fusions were grownin SC-His-Leu to OD₆₀₀=0.5 at 30° C. Cells were normalized to 12 OD₆₀₀units and washed with 10 ml of TE (10 mM Tris pH 7.5; 1 mM EDTA) buffer.Preparation of protein extracts and immunoprecipitation were aspreviously described (30) except for the use of anti-HA.11 monoclonalantibodies (clone 16B12; Covance, Princeton, N.J.) and Protein G-PLUSagarose beads (Santa Cruz Biotechnology; Santa Cruz, Calif.). Sampleswere resolved by SDS-PAGE. The leXA₈₇-66 fusion protein was probed usingmonoclonal antibodies 7E5 which specifically detects p66 (31).

In vitro heterodimerization in the presence of NNRTIs

The heterodimerization of bacterially expressed wild-type p66-His andGST-p51 (or mutants) was assessed in bacterial lysates as describedpreviously (22). To determine the capacity of efavirenz to bind to aparticular RT subunit, 500 μl reactions in lysis buffer (without NP-40)(22), 5 μg of p66-His, 5 μg GST-p51 or no recombinant protein (totalprotein concentration was 0.8 μg/ml in each reaction) were incubatedovernight at 40° C. with increasing concentrations of efavirenz. Lysateswere washed 4 times with lysis buffer using a centricon-YM-50 filterdevice (Millipore Corporation, Bedford, Mass.) to remove unbound drug. 5μg of the corresponding RT subunit was applied to the washed lysates (in500 μl) and incubated for 1.5 hr at 40° C. Heterodimers were capturedonto beads (22), resolved by SDS/PAGE and detected using RT antibodies(monoclonal antibody 5B2) (31).

Results

Enhancement of B-Gal Activity by NNRTIs

To test the effects of NNRTIs on the association of the RT polypeptideswe used a yeast genetic assay that measures RT heterodimerization (22).In this assay yeast expressing the p66 subunit of the HIV-1 RT fused tothe C-terminus of lexA₈₇ (lexA₈₇-66) and the p51 subunit fused to theGal4AD (Gal4AD-51) constitutively interact, resulting in the activationof the expression from an integrated Lac Z reporter gene. To test forthe effects of the NNRTIs on this interaction, 10 drugs representing 7different NNRTI classes were added to the culture medium during growthof the yeast and b-gal levels were determined. Of the 10 NNRTIs tested,9 demonstrated a dramatic concentration-dependent enhancement of b-galactivity compared to cells not treated with drug (FIGS. 7A and 7B). Nosignificant toxicity, as determined by the growth rate, was observed forthe drug concentrations tested compared to untreated controls (resultsnot shown). Efavirenz was the most potent of the compounds, mediating a40-fold increase in b-gal activity at the highest drug concentrationtested (FIG. 7A). The carboxanilide UC781 was the second most potentdrug, followed by UC10 and a quinoxaline, HBY 097 (FIGS. 7A and 7B). Theremainder of the NNRTIs were less potent but still displayed 8-10 foldincreases in b-gal activity at the highest concentrations tested (FIGS.7A and 7B). In contrast delaviridine was devoid of b-gal enhancingactivity (FIG. 7A).

Enhancement of B-Gal Activity by NNRTIs is Specific for RTHeterodimerization

The specificity of the b-gal enhancement by NNRTIs was investigated.Yeast transformed with the empty vectors pSH2-1 and pGADNOT, whichexpress lexA₈7 and Gal4AD, respectively were treated with serialdilutions of the most potent b-gal enhancing drug, efavirenz. Weobserved no increase in b-gal activity even in the presence of 15 μM ofdrug (data not shown). The capacity of efavirenz to enhance b-galactivity of several unrelated protein-protein interaction pairs,including moloney murine leukemia virus reverse transcriptase withelongation factor release factor 1 (M.O., unpublished), was alsoexamined and no enhancement or inhibition of b-gal activity wasobserved.

The Y181C mutation in the p66 subunit of the HIV-1 RT confers more thana 100-fold increase in resistance to nevirapine (17). This mutationdirectly affects drug binding (13, 14). To further establish thespecificity of the b-gal enhancement by NNRTIs, Y181C was introducedinto the plasmid encoding the lexA₈₇-66 fusion protein. Yeast werecotransformed with various pairs of plasmids and grown in the presenceof nevirapine. The presence of the Y181C change in the p66 bait totallynegated the enhancement effect by nevirapine (FIG. 8). In contrast, asignificant level of b-gal enhancement was still retained in thepresence of efavirenz (results not shown), consistent with the very lowlevel of resistance conferred by Y181C to this drug. These data providecompelling evidence that the b-gal enhancement effect is due to aspecific interaction of nevirapine with the p66 subunit of the HIV-1 RT.

NNRTIs can Enhance B-Gal Activity of Dimerization Defective Mutants

Previous studies have shown that the L234A mutation in HIV-1 RTabrogates RT dimerization (22, 32). Other studies on the role of thetryptophan repeat motif (codons 398-414), present in the connectionsubdomains of both subunits, showed that the W401A mutation alsodiminishes RT dimerization in the Y2H assay (G.T. unpublished data). Weinvestigated the effect of the NNRTIs, efavirenz and UC781 on the b-galenhancement effect on these dimerization defective RT mutants.Interestingly, yeast treated with efavirenz and expressing the W401Achange in one or both subunits showed a dramatic increase in b-galactivity compared to no drug (FIG. 9A). b-gal activity in yeastexpressing the W401A mutation in both expressing dimerization defectivemutants to coimmunoprecipitation. Yeast expressing both p66 bait and p51prey fusions containing the W401A mutation (lexA₈₇-66W401A andGal4AD-HA-51W401A) or the L234A change (lexA₈₇-66L234A andGal4AD-HA-51L234A) were grown in the presence of efavirenz (1.6 μM),UC781 (16 μM) or no drug. Hemagglutinin (HA) antibodies were used toimmunoprecipitate the p51 prey and the presence of any bound p66 baitwas then detected using anti-p66 specific antibodies. Forcoimmunoprecipitation of the p66 bait fusions, samples were divided intotwo with one part processed without added NNRTI while the other wasmaintained in drug at the same concentration used during growth ofyeast. Yeast grown in the absence of drug was also processed withoutdrug or in the presence of 1.6 μM of efavirenz. A clear increase in theamount of lexA₈₇-66W401A and lexA₈₇-66L234A associated with the p51 preywas observed for yeast grown in the presence of efavirenz compared to nodrug (FIGS. 4A and 4B). Similar experiments with yeast grown in UC781revealed heterodimer formation for yeast expressing the W401A mutant butnot for the L234A mutant; these data corresponding to the levels ofb-gal activity in the cells (FIGS. 4A and 4B).

No significant difference in the amount of coimmunoprecipitated p66 baitin the absence or presence of drug was observed indicating that theheterodimer was stable under the conditions of the assay. Interestingly,there was significantly more heterodimer present in yeast lysatesobtained from cells grown in the absence of drug to which efavirenz wasadded during the coimmunoprecipitation procedure for the W401A mutant(FIG. 10A). These data suggest that some heterodimer formation couldoccur in vitro. Levels of mutant p66 bait and p51 prey fusions presentin the original lysate from yeast grown in the absence and presence ofdrug were similar indicating that the increase in coimmunoprecipitatedp66 bait in the presence of drug was not due to increased levels offusion proteins. It is clear from these experiments that NNRTIs testeddo act by inducing heterodimerization of p66 and p51 in the Y2H assayand that the increased dimer formation correlates with the increase inb-gal activity.

Efavirenz Enhances the Association of Wild-Type and Mutant p66 and p51in Lysates in Vitro

To explore whether NNRTIs could enhance dimerization in vitro, bacteriallysates containing either p66-His or GST-p51 were prepared and combinedin the presence of increasing concentrations of efavirenz. In theabsence of inhibitor a small amount of dimer was present as indicated bydetectable amounts of p66-His. A concentration dependent increase indimer formation was observed in the presence of increasingconcentrations of efavirenz (FIG. 11). The enhancement effect ofefavirenz on the L234A and W401A mutants was also assessed. Bacteriallysates separately expressing p66L234A-His and GST-p51L234A orp66W401A-His and GST-p51W401A were combined as above and incubated inthe presence of increasing concentrations of efavirenz. A significantincrease in dimer formation was observed in the presence of a 10-foldmolar excess of efavirenz for the W401A mutant (FIG. 11). A 100-foldmolar excess of efavirenz over RT was required to induce detectableenhancement of dimerization of the L234A mutant (FIG. 11). These dataare consistent with the coimmunoprecipitation experiments and indicatethat the enhancement of dimerization by efavirenz is due to its specificinteraction with the HIV-1 RT and not dependent on the fusion proteinsused in the Y2H assay nor on components present in the yeast cells invivo.

Other NNRTIs Enhance Heterodimerization of RT Subunits in Vitro

We extended our in vitro study by testing the remaining NNRTIs for theircapacity to enhance the dimerization of GST-p51 and p66-His in vitro.Consistent with our Y2H data we observed that efavirenz was the mostpotent enhancer of dimerization. The relative in vitro potencies of theother NNRTIs correlated well with their b-gal enhancing effect in yeast(FIGS. 7 and 12). In contrast, UC781 and UC10 were poor dimerizationinducers in bacterial lysates compared with their b-gal enhancingactivities. The low dimerization enhancement activity of these drugs maybe a function of both their poor solubility and the conditions of the invitro assay (which was performed at 4° C.). In contrast, the conditionsof the yeast assay, which was carried out at 30° C. with agitation, mayhave facilitated solubilization of UC781 and UC10. Interestingly,delavirdine was also inactive in vitro indicating that the lack ofeffect in yeast was not a result of the inability of this drug topenetrate the cells.

Efavirenz Enhances Heterodimerization by Binding to p66-His but notGST-p51

To help elucidate the mechanism by which efavirenz enhancesheterodimerization we assessed whether this drug could bind to eitherp66-His or GST-p51. Bacterial lysates expressing p66-His, GST-p51 or norecombinant protein were preincubated in the absence or presence ofincreasing concentrations of efavirenz. Unbound drug was removed fromthe lysates by a series of washes and the presence of any remaining drugwas assayed by the addition of the cognate RT subunit. p66-His andGST-p51 was added to a washed mock bacterial lysate to assess theefficiency of efavirenz removal. When p66-His was preincubated withefavirenz we observed enhancement of dimerization with subsequentlyadded GST-p51 at all drug concentrations (FIG. 13). This enhancement wassimilar to controls where p66-His and GST-p51 were simultaneouslycombined with various drug concentrations (FIG. 12). A 100-foldreduction in the potency of heterodimerization compared to p66-Hispreincubated with efavirenz was observed in the washed mock bacteriallysate (FIG. 13). GST-p51 preincubated with drug, washed and thensubjected to the functional heterodimerization assay displayed the samepattern of heterodimerization observed for the drug-treated mockbacterial lysate. These data indicate that efavirenz binds tightly top66-His but not GST-p51 and that this binding then promotesheterodimerization with subsequent added GST-p51.

Discussion

This study reports a previously undescribed property of certainNNRTIs—their capacity to enhance heterodimerization of the p66 and p51subunits of the HIV-1 RT. This effect was observed both in the Y2Hsystem, detecting dimerization of p66 and p51 using b-gal activity as areadout, and confirmed in coimmunoprecipitation experiments. Thephenomenon was also observed in vitro using bacterially expressedGST-p51 and p66-His showing that it is not specific to yeast. NNRTIswere also able to induce the dimerization of the interaction defectivemutants L234A and W401A. Furthermore, efavirenz can bind tightly top66-His and then subsequently promote heterodimerization. The dataindicate that NNRTIs have properties similar to conventional CIDs intheir capacity to enhance the interaction between two proteins. As theinteraction between p66 and p51 occurs naturally and the effect of theNNRTIs is to enhance this interaction then these small molecules arebest described as chemical enhancers of dimerization.

Correlation Between in Vitro and in Vivo Enhancement ofHeterodimerization by NNRTIs

The most potent b-gal enhancing NNRTIs in the Y2H RT dimerization assaywere efavirenz, UC781 and HBY 097. These drugs are second generationNNRTIs that are also extremely potent inhibitors of HIV-1 replication invitro (21, 25, 27). Efavirenz and UC781 differ from the other NNRTIs inthat they bind very tightly to the RT heterodimer and exhibit very slowdissociation rates (k_(off)) (34, 35). The tight binding properties ofefavirenz and UC781 may in part have contributed to their potency asenhancers of heterodimerization in yeast. There was generally a verygood correlation between the relative potency in inducing dimerizationof the NNRTIs in vitro and in yeast, with the exception of UC781 andUC10.

Relationship Between Drug Induced Enhancement of Dimerization,Structural Changes in the HIV-1 RT and RT Inhibitory Activity.

NNRTIs bind in a hydrophobic pocket at the base of the p66 thumbsubdomain which is proximal to (˜10 Å), but distinct from the polymeraseactive. It is clear that the size of the NNRTI binding pocket is smallcompared to the extensive dimer interface (FIG. 14). No strongcorrelation was found between the extent of the p66/p51 interface (36,37) in the structures of the HIV-1 RT in complex with several NNRTIs andthe drug concentration mediating a 5-fold enhancement of b-gal activity.Thus, the NNRTI effect on heterodimerization is not a simple function ofthe surface area buried at the interface, and NNRTIs may affectdimerization by other mechanisms in addition to modulating the extent ofthe contacts. The position of the drug in the pocket and the degree ofNNRTI interaction with the p51 subunit were found to vary significantlyamong the different RT/NNRTI complexes (FIG. 9), and the changes in thevicinity of the bound NNRTIs may also play a role in heterodimerformation.

Binding of efavirenz to RT is accompanied by conformational changes inthe binding pocket region, and these changes (including at Leu234) (38),may also influence dimer formation. Delavirdine is the longest NNRTIinhibitor and a portion of it protrudes outside the NNRTI binding sitecausing the largest distortion of the p66 subunit of any of the NNRTIsstudied to date (39). Delavirdine binds the furthest away from thep66/p51 interface (closest distance between delavirdine and p51 is 5.1 Åcompared to 3.8 Å for UC781) (FIG. 15) The unique characteristics of theinteraction of delavirdine with HIV-1 RT suggest that this NNRTI maybind to p66 in a distinctive way that does not favor the enhancement ofdimerization.

The relationship between the RT inhibitory activity of the NNRTIs in anexogenous RT assay (50% inhibitory concentration) and the concentrationof drug that mediates a 5-fold increase in b-gal activity in the Y2Hassay was compared. Efavirenz was the most potent in both assays whileUC38 was the least active (results not shown). Examination of the datarevealed a fair correlation (r=0.6) between these two parameterssuggesting a relationship between the b-gal enhancement effect and RTinhibitory activity in vitro.

Potential Mechanisms for NNRTI Enhancement of Dimerization

How might the NNRTIs enhance heterodimerization? One possible modelinvolves NNRTI binding directly to the p66 monomer. Drug binding tomonomeric p66 may stabilize a conformation that is more conducive toheterodimer formation, and a more potent NNRTI may effectively increasethe concentration of p66 in a conformation that promotes dimerization.Alternatively, efavirenz may cause the p66 monomer to have aconformational flexibility that allows this subunit to more readilyundergo structural changes necessary for dimerization. A second modelwould entail NNRTIs binding only to the heterodimer and as a consequencestabilizing the dimer. The binding could shift the equilibrium towardthe dimer. The data suggest that efavirenz binds tightly to p66.However, as bacterially expressed p66 comprises a population of monomersand homodimers it is unclear whether GST-p51 is binding directly tomonomeric p66 complexed with drug or is exchanging with one p66 subunitin the drug bound homodimer. Elucidation of the exact mechanism of NNRTIinduced enhancement of dimerization will require further studies.

The findings may have biological significance in terms of effects onvirus replication. Drug binding to p66 could potentially modulate theinteraction between Pr160^(GagPol) precursors which may affectregulation of HIV-1 protease-specific cleavage of this polyprotein.Further, the Y2H RT dimerization assay can potentially be used to screenfor NNRTIs with the capacity to bind and mediate the appropriateconformational changes in the p66 subunit that results in enhancedbinding to p51. It is possible that novel allosteric inhibitors of RTmay be selected using this assay.

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What is claimed:
 1. A method of determining whether a compound enhancesformation of a complex between a p66 subunit polypeptide of HIV-1reverse transcriptase and a p51 subunit polypeptide of HIV-1 reversetranscriptase which comprises: a) contacting a yeast cell with thecompound, which cell comprises (i) a first plasmid which expresses afusion protein comprising the p66 subunit polypeptide of HIV-1 reversetranscriptase, (ii) a second plasmid which expresses a fusion proteincomprising the psi subunit polypeptide of HIV-1 reverse transcriptase,and (iii) a reporter gene which is activated in the presence of acomplex between the p66 subunit polypeptide and the p51 subunitpolypeptide; b) determining a level of activity of the reporter gene inthe cell in the presence of the compound; and c) comparing the level ofactivity of the reporter gene determined in step (b) with a level ofactivity of the reporter gene determined in the absence of the compound,wherein an increased level of activity of the reporter gene determinedin step (b) compared to the level of activity determined in the absenceof the compound indicates that the compound enhances formation of acomplex between the p51 subunit polypeptide of HIV-1 reversetranscriptase and the p66 subunit polypeptide of HIV-1 reversetranscriptase.
 2. A method of making a pharmaceutical composition whichcompriseS: a) determining whether a compound not previously knownenhances formation of a complex between a p66 subunit polypeptide ofHIV-1 reverse transcriptase and a p51 subunit polypeptide of HIV-1reverse transcriptase by the method of claim 1, b) recovering thecompound if it is determined to enhance formation; and c) admixing thecompound with a pharmaceutically acceptable carrier.
 3. The method ofclaim 1, wherein (a) the fusion protein expressed by the first plasmidcomprises a peptide having a DNA binding domain, and (b) the fusionprotein expressed by the second plasmid comprises a peptide having atranscription activation domain.
 4. The method of claim 3, wherein theDNA binding domain is a LexA DNA binding domain.
 5. The method of claim4, wherein the peptide having a DNA binding domain comprises LexA aminoacid residues 1-87.
 6. The method of claim 4, wherein the peptide havinga DNA binding domain comprises LexA amino acid residues 1-202.
 7. Themethod of claim 3, wherein the DNA binding domain is a GAL4 DNA bindingdomain.
 8. The method of claim 3, wherein the transcription activationdomain is a GAL4 transcription activation domain.
 9. The method of claim8, wherein the peptide having the transcription activation domaincomprises GAL4 amino acid residues 768-881.
 10. The method of claim 3,wherein the transcription activation domain is a VP16 transcriptionactivation domain.
 11. The method of claim 1, wherein (a) the fusionprotein expressed by the first plasmid comprises a peptide having atranscription activation domain, and (b) the fusion protein expressed bythe second plasmid comprises a peptide having a DNA binding domain. 12.The method of claim 11, wherein the DNA binding domain is a LexA DNAbinding domain.
 13. The method of claim 12, wherein the peptide having aDNA binding domain comprises LeXA amino acid residues 1-87.
 14. Themethod of claim 12, wherein the peptide having a DNA binding domaincomprises LexA amino acid residues 1-202.
 15. The method of claim 11,wherein the DNA binding domain is a GAL4 DNA binding domain.
 16. Themethod of claim 11, wherein the transcription activation domain is aGAL4 transcription activation domain.
 17. The method of claim 16,wherein the transcription activation domain comprises GAL4 amino acidresidues 768-881.
 18. The method of claim 11, wherein the transcriptionactivation domain is a VP16 transcription activation domain.
 19. Themethod of claim 1, wherein the fusion protein expressed by the firstplasmid, the second plasmid or both plasmids comprises a peptidecomprising consecutive alanine residues.
 20. The method of claim 19,wherein the peptide comprising consecutive alanine residues comprises atleast 6 alanine residues.
 21. The method of claim 1, wherein the fusionprotein comprises an influenza hemagglutinin (HA) epitope tag.
 22. Themethod of claim 1, wherein the reporter gene is a LacZ reporter gene.23. The method of claim 1, wherein (a) the fusion protein expressed bythe first plasmid comprises a peptide comprising a LexA protein DNAbinding domain, wherein the p66 subunit polypeptide is bound at itsC-terminal amino acid to a N-terminal amino acid of the peptidecomprising a LexA protein DNA binding domain; and (b) the fusion proteinexpressed by the second plasmid comprises a Gal4 peptide correspondingto amino acids 768-881 of Gal4, and an influenza hemagglutiflin (HA)epitope tag, which Gal4 peptide is bound at its C-terminal amino acid toa N-terminal amino, acid of the influenza hemagglutinin (Ha) epitopetag, which influenza hemagglutinin (HA) epitope tag is bound at itsC-terminal amino acid to a N-terminal amino acid of the p51 subunitpolypeptide.
 24. The method of claim 1, wherein (a) the fusion proteinexpressed by the first plasmid comprises a peptide comprising a LexAprotein DNA binding domain, wherein the p66 subunit polypeptide is boundat it's C-terminal amino acid to a N-terminal amino acid of the peptidecomprising a LexA protein DNA binding domain; and (b) the fusion proteinexpressed by the second plasrnid comprises a Gal4 peptide correspondingto amino acids 768-881 of Gal4, which Gal4 peptide is bound at itsC-terminal amino acid to a N-terminal amino acid of the p51 subunitpolypeptide.
 25. The method of claim 1, wherein (a) the fusion proteinexpressed by the first plasmid comprises a LexA peptide corresponding toamino acid residues 1-87, wherein the LexA peptide is bound at itsC-terminal amino acid to a N-terminal amino acid of the of the p66subunit polypeptide; and (b) the fusion protein expressed by the secondplasmid comprises a Gal4 peptide corresponding to amino acids 768-881 ofGal4, and an influenza hemagglutiflin (HA) epitope tag, which Gal4peptide is bound at its C-terminal amino acid to a N-terminal amino acidof the influenza hemagglutinin (HA) epitope tag, which influenzahemagglutinin (HA) epitope tag is bound at its C-terminal amino acid toa N-terminal amino acid of the p51 subunit polypeptide.
 26. The methodof claim 1, wherein (a) the fusion protein expressed by the firstplasmid comprises a LexA peptide corresponding to amino acid residues1-87, wherein the LexA peptide is bound at its C-terminal amino acid toa N-terminal amino acid of the of the p66 subunit polypeptide; and (b)the fusion protein expressed by the second plasmid comprises a Gal4peptide corresponding to amino acids 768-881 of Gal4, which Gal4 peptideis bound at its C-terminal amino acid to a N-terminal amino acid of thep51 subunit polypeptide.
 27. The method of claim 1, wherein (a) thefusion protein expressed by the first plasmid comprises a LexA peptidecorresponding to amino acid residues 1-202, and a peptide comprising sixconsecutive alanine residues, wherein the LexA peptide is bound at itsC-terminal amino acid to a N-terminal amino acid of the peptidecomprising six consecutive alanine residues, wherein the peptidecomprising six consecutive alanine residues is bound at its C-terminalamino acid to a N-terminal amino acid of the p66 subunit polypeptide;and (b) the fusion protein expressed by the second plasmid comprises aGal4 peptide corresponding to amino acids 768-881 of Gal4, which Gal4peptide is bound at its C-terminal amino acid to a N-terminal amino acidof the p51 subunit polypeptide.
 28. The method of claim 1, wherein (a)the fusion protein expressed by the first plasmid comprises a LexApeptide corresponding to amino acid residues 1-202, and a peptidecomprising six consecutive alanine residues, wherein the LexA peptide isbound at its C-terminal amino acid to a N-terminal amino acid of thepeptide comprising six consecutive alanine residues, wherein the peptidecomprising six consecutive alanine residues is bound at its C-terminalamino acid to a N-terminal amino acid of the p66 subunit polypeptide;and (b) the fusion protein expressed by the second plasmid comprises aGal4 peptide corresponding to amino acids 768-881 of Gal4, and aninfluenza hemagglutiflin (HA) epitope tag, which Gal4 peptide is boundat its C-terminal amino acid to a N-terminal amino acid of the influenzahemagglutinin (HA) epitope tag, which influenza hemagglutinin (HA)epitope tag is bound at its C-terminal amino acid to a N-terminal aminoacid of the p51 subunit polypeptide.
 29. The method of claim 1, wherein(a) the fusion protein expressed by the first plasmid comprises a Gal4peptide corresponding to amino acids 768-881 of Gal4, an influenzahemagglutiflin (HA) epitope tag, and a peptide comprising sixconsecutive alanine residues, wherein the Gal4 peptide is bound at itsC-terminal amino acid to a N-terminal amino acid of the influenzahemagglutinin (HA) epitope tag, wherein the influenza hemagglutinin (HA)epitope tag is bound at its C-terminal amino acid to a N-terminal aminoacid of the peptide comprising six consecutive alanine residues, whereinthe peptide comprising six consecutive alanine residues is bound at itsC-terminal amino acid to a N-terminal amino acid of the p66 subunitpolypeptide; and (b) the fusion protein expressed by second plasmidcomprises a peptide comprising a LexA protein DNA binding domain,wherein the p51 subunit polypeptide is bound at its C-terminal aminoacid to a N-terminal amino acid of the peptide comprising a LexA proteinDNA binding domain.
 30. The method of claim 1, wherein (a) the fusionprotein expressed by the first plasmid comprises a Gal4 peptidecorresponding to amino acids 768-881 of Gal4, an influenza hemagglutinin(HA) epitope tag, and a peptide comprising six consecutive alanineresidues, wherein the Gal4 peptide is bound at its C-terminal amino acidto a N-terminal amino acid of the influenza hemagglutinin (HA) epitopetag, wherein the influenza hemagglutinin (HA) epitope tag is bound atits C-terminal amino acid to a N-terminal amino acid of the peptidecomprising six consecutive alanine residues, wherein the peptidecomprising six consecutive alanine residues is bound at its C-terminalamino acid to a N-terminal amino acid of the p66 subunit polypeptide;and (b) the fusion protein expressed by second plasmid comprises apeptide comprising a LexA protein DNA binding domain, wherein peptidecomprising a LexA protein DNA binding domain is bound at its C-terminalamino acid to a N-terminal amino acid of the p51 subunit polypeptide.31. The method of claim 1, wherein (a) the fusion protein expressed bythe first plasmid comprises a Gal4 peptide corresponding to amino acids768-881 of Gal4, an influenza hemagglutinin (HA) epitope tag, and apeptide comprising six consecutive alanine residues, wherein the Gal4peptide is bound at its C-terminal amino acid to a N-terminal amino acidof the influenza hemagglutinin (HA) epitope tag, wherein the influenzahemagglutinin (HA) epitope tag is bound at its C-terminal amino acid toa N-terminal amino acid of the peptide comprising six consecutivealanine residues, wherein the peptide comprising six consecutive alanineresidues is bound at its C-terminal amino acid to the a N-terminal aminoacid of the p66 subunit polypeptide; and (b) the fusion proteinexpressed by second plasmid comprises a peptide comprising a Gal4protein DNA binding domain, which peptide comprising a Gal4 protein DNAbinding domain is bound at its C-terminal amino acid to a N-terminalamino acid of the psi subunit polypeptide.
 32. The method of claim Awherein (a) the fusion protein expressed by the first plasmid comprisesa Gal4 peptide corresponding to amino acids 768-881 of Gal4, wherein theGal4 peptide is bound at its C-terminal amino acid to the a N-terminalamino acid of the p66 subunit polypeptide; and (b) the fusion proteinexpressed by second plasmid comprises a peptide comprising a LexAprotein DNA binding domain, wherein the p51 subunit polypeptide is boundat its C-terminal amino acid to a N-terminal amino acid of the peptidecomprising a LexA protein DNA binding domain.
 33. The method of claim 1,wherein (a) the fusion protein expressed by the first plasmid comprisesa Gal4 peptide corresponding to amino acids 768-881 of Gal4, wherein theGal4 peptide is bound at its C-terminal amino acid to a N-terminal aminoacid of the p66 subunit polypeptide; and (b) the fusion proteinexpressed by second plasmid comprises a peptide comprising a LexAprotein DNA binding domain, which peptide comprising a LexA protein-DNAbinding domain is bound at its C-terminal amino acid to a N-terminalamino acid of the p51 subunit polypeptide.
 34. The method of claim 1,wherein (a) the fusion protein expressed by the first plasmid comprisesa Gal4 peptide corresponding to amino acids 768-881 of Gal4, wherein theGal4 peptide is bound at its C-terminal amino acid to a N-terminal aminoacid of the p66 subunit polypeptide; and (b) the fusion proteinexpressed by second plasmid comprises a peptide comprising a Gal4protein DNA binding domain, which peptide comprising a Gal4 protein DNAbinding domain is bound at its C-terminal amino acid to a N-terminalamino acid of the p51 subunit polypeptide.
 35. A method of enhancingformation of a complex between a p51 subunit polypeptide of HIV-1reverse transcriptase and a p66 subunit polypeptide of HIV-1 reversetranscriptase, with a compound not previously known which comprises: a)contacting a yeast cell with the compound, which cell comprises (i) afirst plasmid which expresses a fusion protein comprising the p66subunit polypeptide of HIV-1 reverse transcriptase, (ii) a secondplasmid which expresses a fusion protein comprising the p51 subunitpolypeptide of HIV-1 reverse transcriptase, and (iii) a reporter genewhich is activated in the presence of a complex between the p66 subunitpolypeptide and the p51 subunit polypeptide, and determining a level ofactivity of the reporter gene in the cell in the presence of thecompound; b) comparing the level of activity of the reporter genedetermined in step (a) with a level of activity of the reporter genedetermined in the absence of the compound, wherein an increased leveP ofactivity of the reporter gene determined in step (a) indicates that thecompound enhances formation of a complex between the p51 subunitpolypeptide of HIV-1 reverse transcriptase and the p66 subunitpolypeptide of HIV-1 reverse transcriptase; c) contacting either (1) thep51 subunit polypeptide, (2) the p66 subunit polypeptide, or (3) boththe p51 subunit polypeptide and the p66 subunit polypeptide, with aneffective amount of the compound determined to enhance formation of thecomplex in step (c), so to thereby enhance formation of a complexbetween the p51 subunit polypeptide of HIV-1 reverse transcriptase and ap66 subunit polypeptide of HIV-1 reverse transcriptase.
 36. The methodof claim 35, wherein the p51 subunit polypeptide of HIV-1 reversetranscriptase and the p66 subunit polypeptide of HIV-1 reversetranscriptase are present in a subject and the contacting is effected byadministering the compound to the subject.
 37. The method of claim 36,wherein the compound is administered orally, intravenously,subcutaneously, intramuscularly, topically or by liposome-mediateddelivery.
 38. The method of claim 36, wherein the subject is a humanbeing, a primate, an equine, an avian, a bovine, a porcine, a canine, afeline or a mouse.
 39. The method of claim 36, wherein the effectiveamount of the compound is between about 1 mg and about 50 mg per kg bodyweight of the subject.
 40. The method of claim 39, wherein the effectiveamount of the compound is between about 2 mg and about 40 mg per kg bodyweight of the subject.
 41. The method of claim 40, wherein the effectiveamount of the compound is between about 3 mg and about 30 mg per kg bodyweight of the subject.
 42. The method of claim 41, wherein the effectiveamount of the compound is between about 4 mg and about 20 mg per kg bodyweight of the subject.
 43. The method of claim 42, wherein the effectiveamount of the compound is between about 5 mg and about 10 mg per kg bodyweight of the subject.
 44. The method of claim 43, wherein the compoundis administered at least once per day.
 45. The method of claim 36,wherein the compound is administered daily.
 46. The method of claim 36,wherein the compound is administered every other day.
 47. The method ofclaim 36, wherein the compound is administered every 6 to 8 days. 48.The method of claim 36, wherein the compound is administered weekly.